microRNAs as Biomarkers for Endometriosis

ABSTRACT

Disclosed herein are compositions and methods useful for the diagnosis, assessment, and characterization of endometriosis in a subject in need thereof, based upon the expression level of at least one miRNA that is associated with endometriosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/184,894, filed Feb. 25, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/329,436, filed Feb. 28, 2019, which is a U.S.national phase application filed under 35 U.S.C. § 371 claiming benefitto PCT International Patent Application No. PCT/US2017/049284, filed onAug. 30, 2017, which claims priority to U.S. Provisional Application No.62/381,130, filed Aug. 30, 2016, each of which disclosures isincorporated herein by reference in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The present application hereby incorporates by reference the entirecontents of the text file named “047162-5124_02US_SequenceListing.txt”in ASCII format. The text file containing the Sequence Listing of thepresent application was created on Nov. 1, 2021 and is 8,613 bytes insize.

BACKGROUND

Micro RNAs (miRNAs) are a class of highly conserved small endogenousnoncoding, functional RNA molecules of 19-24 nucleotides; they controlthe translation and stability of targeted RNAs by base-pairing tocomplementary sites and induce repression or degradation of messengerRNA transcripts (Bartel et al., 2009, Cell, 136:215-233). They play apivotal role in the regulation of development and cellular homeostasisof diverse biological processes (Hassan et al., 2015, Bone, 81:746-756).They exist intracellularly as well as in the serum (Corcoran et al.,2011, Clin Chem, 57(1):18-32) and possess specific characteristics thatmake them promising biomarker candidates for the diagnosis of variousdiseases (Sayed et al., 2014, Heart Lung Cir, 23:503-510, Coskun et al.,2012, World J Gastroenterol, 18:4629-4634, Dorval et al., 2013, FrontMol Neurosci, 6:24, Jin et al., 2013, Cell Mol Neurobiol, 5:601-613, Liuet al., 2014, World J Gastroenterol, 20:12007-12017, Ulivi et al., 2014,Molecules, 19:8220-8237, Zhu et al., 2014, Front Genet, 5:149, Bandieraet al., 2015, J Hepatol, 62:448-457). Cell-free miRNAs are stablypresent in several body fluids, including blood serum and plasma, urine,and saliva (Chen et al., 2008, Cell Res, 18:997-1006). Serum miRNAexpression is stable, reproducible, and consistent among individuals.Specific expression patterns have been identified as biomarkers fornumerous diseases including cancers (Schwarzenbach et al., 2014, Nat RevClin Oncol, 11:145-156). miRNAs remain stable as they are released fromcells to the extracellular space in membrane vesicles (Gallo et al.,2012, PLoS One, 7: e30679) or bound to protein complexes (Turchinovichet al., 2011, Nucleic Acids Res, 39:7223-7233, Arroyo et al., 2011, ProcNatl Acad Sci USA, 108:5003-5008). Alterations in miRNA levels in bloodmay reflect changes during normal physiologic processes (Lai et al.,2014, Aging Cell, 13:679-689, Redni et al., 2011, Front Genet, 2:49) andhave been related to several pathologic conditions, includinggynecologic diseases (Zhao et al., 2012, Clin Chem, 58:896-905, Murri etal., 2013, J Clin Endocrinol Metab, 98:E1835-1844).

Endometriosis is a common gynecological disorder, affecting 10% ofreproductive-aged women (Taylor et al., 2011, Reprod Sci,18(9):814-823). It is characterized by the deposition and proliferationof the endometrial cells outside the uterine cavity (Giudice et al.,2010, N Engl J Med, 362:2389-2398, Bulun et al., 2009, N Engl J Med,360:268-279). The major symptoms of endometriosis are pelvic pain in 50%of patients (Eskenazi et al., 1997, Obstet Gynecol Clin North Am,24:235-258) and infertility in 40 to 50% of patients (Ozkan et al.,2008, Ann N Y Acad Sci, 1127:92-100, Moradi et al., 2014, BMC WomensHealth, 14:123). Unfortunately, there are no currently availableaccurate serum biomarkers of this disease. Imaging techniques such asultrasound are unreliable in the diagnosis and staging of endometriosis(Dunselman et al., 2014, Hum Reprod, 29:400-412). Definitive diagnosisof endometriosis is often made only at late stages of the disease bydirect visualization of the lesions with laparascopy and confirmation ofpathology. A major impediment to successful treatment of endometriosisis the failure of diagnosis at an early stage. A simple blood test forendometriosis-specific biomarkers would offer a more timely and accuratediagnosis of the disease and could lead to earlier treatmentintervention. Although there have been considerable efforts to identifysuch biomarkers (Wang et al., 2012, Clin Chem Lab Med, 50:1423-1428, Jiaet al., 2013, Hum Reprod, 28:322-330, Suryawanshi et al., 2013, ClinCancer Res, 19:1213-1224), no clear choice for such noninvasivediagnostic tools has been identified. Serum CA-125 is elevated in somewomen with endometriosis, however it is not specific and has poorsensitivity and specificity. As described above, miRNAs have beencarefully evaluated as biomarkers for several diseases; they holdpromise for a diagnostic marker of endometriosis.

Despite the fact that endometriosis is present in 10% of allreproductive-aged women, and in 20-50% in infertile women, there is nodefinite diagnostic biomarker for endometriosis yet available. Imagingtechniques, such as ultrasound and magnetic resonance imaging, have beenshown to be unreliable in the diagnosis or staging of this disease. Thedirect visualization of lesions and histologic confirmation throughsurgical procedures are currently essential for the definitive diagnosisof endometriosis, which requires general anesthesia, developed surgicalskills, procedural costs, and the risk of potential complications.Therefore, development of new noninvasive diagnostic markers forendometriosis is crucial for early diagnosis and proper treatment andmanagement of the disease. Thus there is a need in the art for improvedcompositions and methods for noninvasive biomarkers of endometriosis.The present disclosure satisfies this unmet need.

SUMMARY

This disclosure provides novel methods of detecting, diagnosing, and/orprognosing endometriosis in female subjects suspected of havingendometriosis, as well as novel methods of treating endometriosis. Manyof the methods provided herein generally relate to detecting miRNAbiomarkers in samples such as blood, serum, or saliva. As such, themethods may be used in non-invasive or minimally-invasive assays todetect endometriosis and may help many female patients avoid moreinvasive procedures such as biopsies of endometrial tissue. The methodsmay also promote early diagnosis and may promote treatment ofendometriosis at an early stage, before the patient has developed moresevere or serious symptoms.

In one aspect, this disclosure provides a method of diagnosing a subjectsuspected of having endometriosis comprising: (a) providing a saliva,sputum, urine, lymphatic fluid, synovial fluid, cerebrospinal fluid,stool, or mucus sample from the subject, wherein the saliva, sputum,urine, lymphatic fluid, synovial fluid, cerebrospinal fluid, stool, ormucus sample comprises miRNA associated with endometriosis; (b)detecting a level of the miRNA associated with endometriosis; (c)comparing the detected level of miRNA associated with endometriosis witha reference value in order to determine a relative level of miRNAassociated with endometriosis in the saliva, sputum, urine, lymphaticfluid, synovial fluid, cerebrospinal fluid, stool, or mucus sample; and(d) diagnosing the subject with endometriosis based on the relativelevel of miRNA associated with endometriosis in the saliva, sputum,urine, lymphatic fluid, synovial fluid, cerebrospinal fluid, stool, ormucus sample, wherein the diagnosing the subject with endometriosis hasa specificity or sensitivity greater than 90%.

In some embodiments, the sample is a fluid. In other embodiments, thesample is blood, plasma, or serum. In other embodiments the sample issaliva. In other embodiments, the sample is a cell-free sample.

In other embodiments, the miRNA is selected from the group consisting ofmiR-125, miR-451, and miR-3613. In other embodiments, the miRNA ismiR-125b. In other embodiments, the miRNA is miR-125b-5p. In otherembodiments, the miRNA is miR-451a. In other embodiments, the miRNA ismiR-3613-5p. In some embodiments, the at least one miRNA is let-7,let-7a, let-7b, let-7b-3p, let-7b-5p, let-7c, let-7d, let-7e, let-7f, orlet-7g. In some embodiments, the miRNA is at least two of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In someembodiments, the reference value is the presence of the at least onemiRNA in samples from subjects that do not have endometriosis.

In another aspect, this disclosure provides a method of detecting miRNAcomprising: (a) providing a saliva, sputum, urine, lymphatic fluid,synovial fluid, cerebrospinal fluid, stool, or mucus sample from asubject, wherein the sample comprises nucleic acids and wherein thesubject is suspected of having endometriosis; (b) performing anamplification or sequencing reaction on the nucleic acids in order todetect a presence of at least one miRNA in the sample, wherein the atleast one miRNA is selected from the group consisting of miR-18,miR-125, miR-126, miR-143, miR-145, miR-150, miR-214, miR-342, miR-451,miR-500, miR-553, miR-3613, miR-4668, and miR-6755; and (c) comparingthe presence of the miRNA to a reference value.

In some embodiments, the sample is a fluid. In other embodiments, thesample is blood, plasma, or serum. In other embodiments the sample issaliva. In other embodiments, the sample is cell-free.

In other embodiments, the miRNA is selected from the group consisting ofmiR-125, miR-451, and miR-3613. In other embodiments, the miRNA ismiR-125b. In other embodiments, the miRNA is miR-125b-5p. In otherembodiments, the miRNA is miR-451a. In other embodiments, the miRNA ismiR-3613-5p. In some embodiments, the miRNA is at least two of thefollowing: miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In someembodiments, the reference value is the presence of the at least onemiRNA in samples from subjects that do not have endometriosis.

In yet another aspect, the disclosure provides for a method comprising:(a) providing a sample from a subject, wherein the sample comprisesnucleic acids and wherein the subject is suspected of havingendometriosis; (b) performing a sequencing reaction on the nucleic acidsto detect a presence of at least one miRNA in the sample, wherein the atleast one miRNA is selected from the group consisting of miR-18,miR-125, miR-126, miR-143, miR-145, miR-150, miR-214, miR-342, miR-451,miR-500, miR-553, miR-3613, miR-4668, and miR-6755; and (c) comparingthe presence of the miRNA to a reference value. In some embodiments, theat least one miRNA is detected by high-throughput or massively-parallelsequencing. In some embodiments, the miRNA is at least one of thefollowing, at least two of the following, or all of the following:miR-125b-5p, miR-451a, and miR-3613-5p. In some embodiments, the miRNAis at least one of the following or all of the following: miR-125b-5p,let-7b, miR-150, miR-342, and miR-3613. In some embodiments, the miRNAis at least two of the following: miR-125b-5p, let-7b, miR-150, miR-342,and miR-3613.

In yet another aspect, the disclosure provides for a method ofdiagnosing a subject exhibiting symptoms of endometriosis or suspectedof having endometriosis, the method comprising: (a) detecting a presenceof at least one miRNA that is not let-7, miR-135, miR-449a, miR-34c,miR-200a, miR-200b, miR-141, miR-125b-5p, miR-150-5p, miR-342-3p,miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-6755-3p,or miR-3613-5p; and (b) diagnosing the subject with endometriosis, whenthe detected miRNA level is above a threshold level, wherein thediagnosing the subject with endometriosis has a specificity orsensitivity greater than 90%. In some embodiments, the disclosureprovides for a method comprising: (a) detecting a presence of at leastone miRNA that is not let-7, let-7a, let-7b, let-7c, let-7d, let-7e,let-7f, let-7g, miR-17-5p, miR-20a, miR-22, miR-135a, miR-135b, miR-135,miR-449a, miR-34c, miR-199a, miR-122, miR-145, miR-141, miR-542-3p,miR-9, miR-200a, miR-200a-3p, miR-200b-3p, miR-200b, miR-141,miR-141-3p, miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-6755-3p, or miR-3613-5p.

In some embodiments, the miRNA is cell-free. In some embodiments, themiRNA is present in a cell-free sample, such as cell-free plasma, serum,saliva and/or urine.

In some embodiments, the diagnosing the subject with endometriosis isdiagnosing the subject with early stage endometriosis. In otherembodiments, the diagnosing the subject with endometriosis is diagnosingthe subject with moderate-to-severe disease. In other embodiments, thediagnosing the subject with endometriosis is diagnosing the subject withstage III or Stage IV endometriosis. In other embodiments, thediagnosing the subject with endometriosis further comprises monitoringthe subject for changes in miRNA expression levels following treatment.

In other embodiments, the diagnosing the subject with endometriosis hasa specificity or sensitivity greater than 75%, greater than 80%, greaterthan 85%, greater than 90%, greater than 95%, or greater than 99%. Inother embodiments, the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%.

In some embodiments, the method further comprises treating the subjectfor endometriosis. In other embodiments, treatment is a medication torelieve pain. In other embodiments, treatment is hormone therapy. Inother embodiments, treatment is a hormonal contraceptive. In otherembodiments, treatment is a Gonadotropin-releasing hormone agonist. Inother embodiments, treatment is a Gonadotropin-releasing hormoneantagonist.

In some embodiments, the detecting comprises: (i) amplifying miRNAwithin the sample by performing a reverse transcription assay using aprimer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with a probe specific for the at least one miRNA, wherein the probeemits a signal upon binding the produced cDNA; and (iii) using adetector to detect the signal emitted by the probe.

In a further aspect, the disclosure provides for a method of detectingmiRNA comprising: (a) providing a sample from a subject, wherein thesample comprises nucleic acids and wherein the subject is suspected ofhaving endometriosis; (b) performing an amplification, microarray orsequencing reaction on the nucleic acids; (c) detecting a presence of atleast one miRNA in the sample, wherein the at least one miRNA isselected from the group consisting of miR-126, miR-214, miR-553, andmiR-4668; and (d) comparing the presence of the miRNA to a referencevalue. In some embodiments, the sample is a fluid. In other embodiments,the sample is blood, plasma, saliva, or serum. In some embodiments, thereference value is the presence of the at least one miRNA in samplesfrom subjects that do not have endometriosis.

In some embodiments, the detecting is by polymerase chain reaction(PCR). In other embodiments, the detecting is by quantitative PCR. Insome embodiments, the detecting is by real-time PCR. In otherembodiments, the detecting comprises hybridizing a unique primer to theat least one miRNA or to cDNA derived from the at least one miRNA. Inother embodiments the detecting further comprises performing a reversetranscription reaction on the at least one miRNA using at least oneprimer or probe specific for the at least one miRNA or using at leastone universal primer. In other embodiments, the detecting comprises: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with an intercalating dye that emits a signal; and (iii)using a detector to detect the emitted signal over time. In otherembodiments, the detecting comprises: (i) amplifying miRNA within thesample by performing a reverse transcription assay using a primer thatspecifically binds the at least one miRNA or using a universal primer,thereby producing cDNA; (ii) contacting the produced cDNA with a probespecific for the at least one miRNA, wherein the probe emits a signalupon binding the produced cDNA; and (iii) using a detector to detect thesignal emitted by the probe. In other embodiments, the probe is attachedto a fluorophore and a quencher. In other embodiments the detecting isby sequencing.

In some embodiments, the endometriosis is diagnosed when the at leastone miRNA is at least 2-fold greater than the reference value. In otherembodiments, the endometriosis is diagnosed when the at least one miRNAis at least 2-fold less than the reference value. In other embodiments,the endometriosis is diagnosed when miRNA-125b-5p is upregulated,miR-150-5p is upregulated, miR-342-3p is upregulated, miR-145-5p isupregulated, miR-143-3p is upregulated, miR-500a-3p is upregulated, ormiR-18a-5p is upregulated. In other embodiments, the endometriosis isdiagnosed when at least three miRNA are upregulated wherein the at leastthree miRNA are from the group consisting of miRNA-125b-5p, miR-150-5p,miR-342-3p, miR-145-5p, miR-143-3p, miR-500a-3p, and miR-18a-5p. Inother embodiments, the endometriosis is diagnosed when miR-6755-3p isdownregulated or miR-3613-5p is downregulated. In other embodiments, theendometriosis is diagnosed when miR-125b-5p is upregulated, miR-451a isupregulated, and miR-3613-5p is downregulated. In some embodiments, theendometriosis is diagnosed when miR-125b-5p is upregulated. In someembodiments, the endometriosis is diagnosed when let-7b is upregulated.

In some embodiments, the subject is negative for the presence of a KRASvariant allele. In other embodiments, the detected miRNA level is usedto determine severity of disease. In other embodiments, the subject is ahuman subject.

In yet a further aspect, the disclosure provides a method of treating asubject with endometriosis, comprising administering an endometriosistreatment to a subject identified as having a differential level of atleast one miRNA selected from the group consisting of miR-18a-5p,miR-125, miR-126, miR-143, miR-145, miR-150-5p, miR-214, miR-342,miR-451, miR-500, miR-553, miR-3613, miR-4668, and miR-6755 in abiological sample of the subject as compared to a comparator. In someembodiments, the method further comprises diagnosing the subject withendometriosis when the subject is identified as having a differentiallevel of at least one miRNA selected from the group consisting ofmiR-18a-5p, miR-125, miR-126, miR-143, miR-145, miR-150-5p, miR-214,miR-342, miR-451, miR-500, miR-553, miR-3613, miR-4668, and miR-6755, ascompared to the comparator.

In some embodiments, the diagnosing the subject with endometriosiscomprises providing a biological sample from the subject and detectingin the biological sample the differential level of at least one miRNAselected from the group consisting of the miR-18a-5p, miR-125, miR-126,miR-143, miR-145, miR-150-5p, miR-214, miR-342, miR-451, miR-500,miR-553, miR-3613, miR-4668, and miR-6755, as compared to thecomparator. In some embodiments, the at least one miRNA is at least oneof the following, at least two of the following, or all of thefollowing: miR-125b-5p, miR-451a, and miR-3613-5p. In some embodiments,the at least one miRNA is at least one of the following or all of thefollowing: miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In someembodiments, the at least one miRNA is at least two of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613.

In some embodiments, the biological sample is a body fluid. In someembodiments, the body fluid is blood, plasma, or serum. In someembodiments, the body fluid is saliva. In some embodiments, the bodyfluid is urine.

In some embodiments, the diagnosing the subject with endometriosis isdiagnosing the subject with early stage endometriosis. In otherembodiments, the diagnosing the subject with endometriosis is diagnosingthe subject with moderate-to-severe disease. In other embodiments, thediagnosing the subject with endometriosis is diagnosing the subject withstage III or stage IV endometriosis. In other embodiments, thediagnosing the subject with endometriosis further comprises monitoringthe subject for changes in miRNA expression levels following treatment.

In some embodiments, the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%. In other embodiments, thetreatment is a medication to relieve pain. In other embodiments, thetreatment is hormone therapy. In other embodiments, treatment isselected from the group consisting of a hormonal contraceptive, aGonadotropin-releasing hormone agonist, and a Gonadotropin-releasinghormone antagonist.

In some embodiments, the method further comprises detecting the at leastone miRNA by performing a polymerase chain reaction (PCR). In someembodiments, the method further comprises detecting the at least onemiRNA by performing quantitative PCR. In some embodiments, the methodfurther comprises detecting the at least one miRNA by: (i) amplifyingmiRNA within the sample by performing a reverse transcription assayusing a primer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with an intercalating dye that emits a signal; and (iii) using adetector to detect the emitted signal over time. In other embodiments,the method further comprises detecting the at least one miRNA by: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with a probe specific for the at least one miRNA, whereinthe probe emits a signal upon binding the produced cDNA; and (iii) usinga detector to detect the signal emitted by the probe. In otherembodiments, the method further comprises detecting the at least onemiRNA by sequencing. In other embodiments, the method further comprisesdetecting the at least one miRNA by high-throughput sequencing ormassively parallel sequencing.

In some embodiments, the at least one miRNA is present at a level atleast 2-fold greater than the reference value. In some embodiments, thesubject is negative for the presence of KRAS variant allele. In someembodiments the subject is a human subject. In some embodiments, a levelof the at least one miRNA is used to determine severity of disease.

In some embodiments, the endometriosis is diagnosed when miRNA-125b-5pis upregulated, miR-150-5p is upregulated, miR-342-3p is upregulated,miR-145-5p is upregulated, miR-143-3p is upregulated, miR-500a-3p isupregulated, or miR-18a-5p is upregulated. In other embodiments, theendometriosis is diagnosed when at least three miRNA are upregulatedwherein the at least three miRNA are from the group consisting ofmiRNA-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, and miR-18a-5p. In other embodiments, the endometriosis isdiagnosed when miR-6755-3p is downregulated or miR-3613-5p isdownregulated. In other embodiments, the endometriosis is diagnosed whenmiR-125b-5p is upregulated, miR-451a is upregulated, and miR-3613-5p isdownregulated. In other embodiments the endometriosis is diagnosed whenmiR-125b-5p is upregulated.

In a further aspect, the disclosure provides a method of diagnosing andtreating a subject suspected of having endometriosis, comprising: (a)providing a saliva, sputum, urine, lymphatic fluid, synovial fluid,cerebrospinal fluid, stool, or mucus sample from the subject, whereinthe sample comprises miRNA associated with endometriosis; (b) detectinga level of the miRNA associated with endometriosis; (c) comparing thedetected level of miRNA associated with endometriosis with a referencevalue in order to determine a relative level of miRNA associated withendometriosis in the fluid sample; (d) diagnosing the subject withendometriosis based on the relative level of miRNA associated withendometriosis in the saliva, sputum, urine, lymphatic fluid, synovialfluid, cerebrospinal fluid, stool or mucus sample, wherein thediagnosing the subject with endometriosis has a sensitivity orspecificity greater than 90%; and (e) administering a treatment to thesubject diagnosed with endometriosis.

In some embodiments, the sample from the subject is a saliva sample. Insome cases, the sample from the subject is a urine sample. In somecases, the sample from the subject is a mucus sample.

In some embodiments, the diagnosing the subject with endometriosis isdiagnosing the subject with early stage endometriosis. In otherembodiments, the diagnosing the subject with endometriosis is diagnosingthe subject with moderate-to-severe disease. In other embodiments, thediagnosing the subject with endometriosis is diagnosing the subject withstage III or stage IV endometriosis. In other embodiments, thediagnosing the subject with endometriosis further comprises monitoringthe subject for changes in miRNA expression levels following treatment.

In some embodiments, the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%. In other embodiments, thetreatment is a medication to relieve pain. In other embodiments, thetreatment is hormone therapy. In other embodiments, treatment isselected from the group consisting of a hormonal contraceptive, aGonadotropin-releasing hormone agonist, and a Gonadotropin-releasinghormone antagonist.

In some embodiments, the method further comprises detecting the at leastone miRNA by performing a polymerase chain reaction (PCR). In someembodiments, the method further comprises detecting the at least onemiRNA by performing quantitative PCR. In some embodiments, the methodfurther comprises detecting the at least one miRNA by: (i) amplifyingmiRNA within the sample by performing a reverse transcription assayusing a primer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with an intercalating dye that emits a signal; and (iii) using adetector to detect the emitted signal over time. In other embodiments,the method further comprises detecting the at least one miRNA by: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with a probe specific for the at least one miRNA, whereinthe probe emits a signal upon binding the produced cDNA; and (iii) usinga detector to detect the signal emitted by the probe. In otherembodiments, the method further comprises detecting the at least onemiRNA by sequencing. In other embodiments, the method further comprisesdetecting the at least one miRNA by high-throughput sequencing ormassively parallel sequencing.

In some embodiments, the at least one miRNA is present at a level atleast 2-fold greater than the reference value. In some embodiments, thesubject is negative for the presence of KRAS variant allele. In someembodiments the subject is a human subject. In some embodiments, a levelof the at least one miRNA is used to determine severity of disease.

In some embodiments, the endometriosis is diagnosed when miRNA-125b-5pis upregulated, miR-150-5p is upregulated, miR-342-3p is upregulated,miR-145-5p is upregulated, miR-143-3p is upregulated, miR-500a-3p isupregulated, or miR-18a-5p is upregulated. In other embodiments, theendometriosis is diagnosed when at least three miRNA are upregulatedwherein the at least three miRNA are from the group consisting ofmiRNA-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, and miR-18a-5p. In other embodiments, the endometriosis isdiagnosed when miR-6755-3p is downregulated or miR-3613-5p isdownregulated. In other embodiments, the endometriosis is diagnosed whenmiR-125b-5p is upregulated, miR-451a is upregulated, and miR-3613-5p isdownregulated. In other embodiments the endometriosis is diagnosed whenmiR-125b-5p is upregulated.

In a further aspect, the disclosure provides a method of diagnosing andtreating a subject suspected of having endometriosis, the methodcomprising: (a) detecting a presence of at least one miRNA that is notlet-7, miR-449a, miR-34c, miR-200a, miR-200b, miR-150, miR-18a, ormiR-141; (b) diagnosing the subject with endometriosis, when thedetected miRNA level is above a threshold level, wherein the diagnosingthe subject with endometriosis has a specificity or sensitivity greaterthan 90%; and (c) administering a treatment to the subject diagnosedwith endometriosis.

In some embodiments, the at least one miRNA comprises miR-125b-5p,miR-451a, and miR-3613-5p. In other embodiments, the at least one miRNAis miR-125b-5p. In other embodiments the at least one miRNA ismiR-3613-5p. In other embodiments, the at least one miRNA is cell-freemiRNA. In other embodiments, the at least one miRNA is present in atleast a 2-fold change compared to the comparator.

In some embodiments, the diagnosing the subject with endometriosis isdiagnosing the subject with early stage endometriosis. In otherembodiments, the diagnosing the subject with endometriosis is diagnosingthe subject with moderate-to-severe disease. In other embodiments, thediagnosing the subject with endometriosis is diagnosing the subject withstage III or stage IV endometriosis. In other embodiments, thediagnosing the subject with endometriosis further comprises monitoringthe subject for changes in miRNA expression levels following treatment.

In some embodiments, the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%. In other embodiments, thetreatment is a medication to relieve pain. In other embodiments, thetreatment is hormone therapy. In other embodiments, treatment isselected from the group consisting of a hormonal contraceptive, aGonadotropin-releasing hormone agonist, and a Gonadotropin-releasinghormone antagonist.

In some embodiments, the method further comprises detecting the at leastone miRNA by performing a polymerase chain reaction (PCR). In someembodiments, the method further comprises detecting the at least onemiRNA by performing quantitative PCR. In some embodiments, the methodfurther comprises detecting the at least one miRNA by: (i) amplifyingmiRNA within the sample by performing a reverse transcription assayusing a primer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with an intercalating dye that emits a signal; and (iii) using adetector to detect the emitted signal over time. In other embodiments,the method further comprises detecting the at least one miRNA by: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with a probe specific for the at least one miRNA, whereinthe probe emits a signal upon binding the produced cDNA; and (iii) usinga detector to detect the signal emitted by the probe. In otherembodiments, the method further comprises detecting the at least onemiRNA by sequencing. In other embodiments, the method further comprisesdetecting the at least one miRNA by high-throughput sequencing ormassively parallel sequencing. In some embodiments, the at least onemiRNA is present at a level at least 2-fold greater than the referencevalue. In some embodiments, the subject is negative for the presence ofKRAS variant allele. In some embodiments the subject is a human subject.In some embodiments, a level of the at least one miRNA is used todetermine severity of disease.

In some embodiments, the endometriosis is diagnosed when miRNA-125b-5pis upregulated, miR-150-5p is upregulated, miR-342-3p is upregulated,miR-145-5p is upregulated, miR-143-3p is upregulated, miR-500a-3p isupregulated, or miR-18a-5p is upregulated. In other embodiments, theendometriosis is diagnosed when at least three miRNA are upregulatedwherein the at least three miRNA are from the group consisting ofmiRNA-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, and miR-18a-5p. In other embodiments, the endometriosis isdiagnosed when miR-6755-3p is downregulated or miR-3613-5p isdownregulated. In other embodiments, the endometriosis is diagnosed whenmiR-125b-5p is upregulated, miR-451a is upregulated, and miR-3613-5p isdownregulated. In other embodiments the endometriosis is diagnosed whenmiR-125b-5p is upregulated.

In another aspect, the disclosure provides a method of diagnosingendometriosis in a subject. In some embodiments, the method comprisesdetermining the level of at least one miRNA in a biological sample ofthe subject, wherein the miRNA is at least one selected from the groupconsisting of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500,miR-451, miR-18, miR-214, miR-126, miR-6755, miR-3613, miR-553, andmiR-4668; and comparing the level of the at least one miRNA in thebiological sample with the level of the at least one miRNA in acomparator, wherein when the level of the at least one miRNA in thebiological sample is different than the level of the at least one miRNAin the comparator, the subject is diagnosed with endometriosis. In someembodiments, the method further comprises the step of treating thesubject for endometriosis.

In some embodiments, the at least one miRNA is the combination ofmiR-125, miR-451 and miR-3613.

In some embodiments, the subject is human.

In some embodiments, the comparator is at least one comparator selectedfrom the group consisting of a positive control, a negative control, anormal control, a wild-type control, a historical control, and ahistorical norm.

In some embodiments, the disclosure provides a method of diagnosingendometriosis in a subject, wherein the method comprises detecting thatthe level of at least one of miR-125, miR-150, miR-342, miR-145,miR-143, miR-500, miR-451, miR-18, miR-214, and miR-126 is increased inthe biological sample compared to the level in the comparator. Inanother embodiment, the disclosure provides a method of diagnosingendometriosis in a subject, wherein the method comprises detecting thatthe level of at least one of miR-6755, miR-3613, miR-553, and miR-4668are decreased in the biological sample compared to the level in thecomparator.

In some embodiments, determining the level of the at least one miRNAutilizes at least one technique selected from the group consisting ofreverse transcription, PCR, and a microarray.

In some embodiments, the biological sample is selected from the groupconsisting of blood, serum, plasma and any combination thereof.

In another aspect, the disclosure also provides a method of diagnosingor providing a prognosis for endometriosis in a subject, methodcomprising the step of: detecting altered expression of at least onegene selected from the group consisting of miR-125, miR-150, miR-342,miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755,miR-3613, miR-553, and miR-4668 in a biological sample of the subjectsuspected of or having endometriosis.

In some embodiments, the at least one gene is the combination ofmiR-125, miR-451 and miR-3613.

In some embodiments, the biological sample is selected from the groupconsisting of blood, serum, plasma, and any a combination thereof.

The disclosure also provides a kit comprising a reagent that selectivelybinds to at least one miRNA, wherein the at least one miRNA is at leastone selected from the group consisting of miR-125, miR-150, miR-342,miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755,miR-3613, miR-553, and miR-4668. In some embodiments, the kit comprisesat least three reagents, wherein the first reagent selectively binds tomiR-125, wherein the second reagent selectively binds to miR-451, andwherein the third reagent selectively binds to miR-3613.

In another aspect, the disclosure provides a method of treating asubject with endometriosis, comprising administering an endometriosistreatment to a subject identified as having a differential level of atleast one selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668 in a biological sample of thesubject as compared to a comparator.

In another aspect, the disclosure provides a method of monitoring aresponse to an endometriosis treatment in a subject being treated forendometriosis, the method comprises determining the level of at leastone miRNA in a biological sample of the subject, wherein the miRNA is atleast one selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668, and comparing the level ofthe at least one miRNA in the biological sample with the level of the atleast one miRNA in a comparator.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of thedisclosure will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the disclosure, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1 is an image depicting the results of differentially expressedmiRNAs in the Microarray Analysis.

FIG. 2 comprising FIG. 2A and FIG. 2B, is a set of images depicting theresults of differentially expressed miRNAs in the endometriosis andcontrol groups as determined by real time quantitative PCR. p-valuesbased on analysis using the Mann-Whitney U test.

FIG. 3 comprising FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D is a set ofimages depicting the result of AUC values of the differentiallyexpressed miRNAs. FIG. 3A, is an image illustrating receiver operatingcharacteristic (ROC) curve analysis of serum miR-125b-5p, miR-150-5p,and miR-342-3p. FIG. 3B, is an image illustrating ROC curve analysis ofserum miR-451a, miR-500a-3p, miR-143-3p. FIG. 3C, is an imageillustrating ROC curve analysis of serum miR-18a-5p, miR-6755-3p, andmiR-3613-5p. miR-125b-5p (FIG. 3A) showed the highest AUC of 0.974. FIG.3D, is an image illustrating that the combination of serum miR-125-b-5p,miR-451a, and miR-3613-5p showed the highest AUC value of 1.000

FIG. 4 is an image depicting the result of relative expression of miRNAsin endometriosis and groups with treatment in baboons as determined byreal time quantitative PCR. p-values based on analysis using theMann-Whitney U test.

FIG. 5 is an image depicting the result of relative expression of miRNAsin saliva in endometriosis and control groups.

FIG. 6 is an image depicting the result of relative expression of miRNAsin saliva in endometriosis and control groups.

FIG. 7 is an image depicting expression levels of miRNA in serum ofwomen who underwent laparoscopy or laparotomy that either confirmedpresence of endometriosis (disease group) or revealed other benignpathology (control group).

DETAILED DESCRIPTION

The present disclosure relates to the discovery that the expressionlevel of particular microRNAs (miRNAs) is associated with endometriosis,such as endometriosis during the proliferative phase. Thus, in variousembodiments described herein, the methods of the disclosure relate tomethods of diagnosing a subject as having endometriosis, methods ofassessing a subject's risk of having or developing endometriosis,methods of assessing the severity of a subject's endometriosis, methodsof stratifying a subject having endometriosis for assignment in aclinical trial, and methods of monitoring endometriosis treatment in asubject. Thus, the disclosure relates to compositions and methods usefulfor the detection and quantification of miRNAs for the diagnosis,assessment, and characterization of endometriosis in a subject in needthereof, based upon the expression level of at least one miRNA that isassociated with endometriosis. The markers of the disclosure can be usedto screen, diagnose, monitor the onset, monitor the progression, andassess the treatment of endometriosis. The markers of the disclosure canbe used to establish and evaluate treatment plans.

In some embodiments, the miRNAs that are associated with endometriosisis a marker or biomarker of endometriosis. In various embodiments, thebiomarkers of the disclosure include one or more of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668. In some embodiments, thebiomarkers of the disclosure include the combination of miR-125b,miR-451, and miR-3613. In some embodiments, the biomarkers of thedisclosure comprise at least two of the following: miR-125b-5p, let-7b,miR-150, miR-342, and miR-3613. In some embodiments, the biomarkers ofthe disclosure comprise at least one of the following, at least two ofthe following, or all of the following: miR-125b-5p, miR-451a, andmiR-3613-5p. In some embodiments, the biomarkers are at least one of thefollowing, at least two of the following, or all of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In some cases, thebiomarkers may further comprise miR-451.

In some embodiments, the miRNAs that are associated with endometriosisis a marker or biomarker of endometriosis. In various embodiments, thebiomarkers of the disclosure include one or more of miR-125b-5p,miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a,miR-18a-5p, miR-214-3p, miR-126-3p, miR-6755-3p, miR-3613-5p, miR-553,and miR-4668-3p. In some embodiments, the biomarkers of the disclosureinclude the combination of miR-125b-5p, miR-451a, and miR-3613-5p. Insome embodiments, the biomarkers include at least two of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In someembodiments, the biomarkers include at least one of the following, atleast two of the following, or all of the following: miR-125b-5p,miR-451a, and miR-3613-5p. In some embodiments, the biomarkers includeat least one of the following, at least two of the following, or all ofthe following: miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. Thebiomarkers may, in some instances, further include let-7b.

In some embodiments, the disclosure provides a marker that predicts anindividual's risk of developing endometriosis. In some embodiments, themarkers of the disclosure can predict risk at a time when a prophylactictherapy can be administered such that the emergence of the disease isprevented.

In some embodiments, the markers of the disclosure are noninvasivebiomarkers for endometriosis that allow for early detection of thedisease without surgical procedures. For example, altered expression ofspecific miRNAs in the biological sample of the subject withendometriosis may correlate with other clinical parameters, such aspelvic pain, infertility, and disease recurrence. Therefore, the markersof the disclosure can be used, not only as biomarkers of the disease,but also as markers for prognosis and recurrence. This is an advantagebecause repeated surgical procedures used in the art for diagnosingendometriosis and related complications can be avoided.

The present disclosure provides biomarkers for the diagnosis andprognosis of endometriosis. Generally, the methods of this disclosurefind use in diagnosing or for providing a prognosis for endometriosis bydetecting the expression levels of biomarkers, which are differentiallyexpressed (up- or down-regulated) in blood, plasma or serum from apatient. Similarly, these markers can be used to diagnose reducedfertility in a patient with endometriosis or to provide a prognosis fora fertility trial in a patient suffering from endometriosis. The presentdisclosure also provides methods of identifying a compound for treatingor preventing endometriosis. The present disclosure provides kits forthe diagnosis or prognosis of endometriosis.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“Antisense,” as used herein, refers to a nucleic acid sequence which iscomplementary to a target sequence, such as, by way of example,complementary to a target miRNA sequence, including, but not limited to,a mature target miRNA sequence, or a sub-sequence thereof. Typically, anantisense sequence is fully complementary to the target sequence acrossthe full length of the antisense nucleic acid sequence.

The term “body fluid” or “bodily fluid” as used herein refers to anyfluid from the body of an animal. Examples of body fluids include, butare not limited to, plasma, serum, blood, lymphatic fluid, cerebrospinalfluid, synovial fluid, urine, saliva, mucous, phlegm and sputum. A bodyfluid sample may be collected by any suitable method. The body fluidsample may be used immediately or may be stored for later use. Anysuitable storage method known in the art may be used to store the bodyfluid sample: for example, the sample may be frozen at about −20° C. toabout −70° C. Suitable body fluids are acellular fluids. “Acellular”fluids include body fluid samples in which cells are absent or arepresent in such low amounts that the miRNA level determined reflects itslevel in the liquid portion of the sample, rather than in the cellularportion. Such acellular body fluids are generally produced by processinga cell-containing body fluid by, for example, centrifugation orfiltration, to remove the cells. Typically, an acellular body fluidcontains no intact cells however, some may contain cell fragments orcellular debris. Examples of acellular fluids include plasma or serum,or body fluids from which cells have been removed.

As used herein, the term “cell-free” refers to the condition of thenucleic acid as it appeared in the body directly before the sample isobtained from the body. For example, nucleic acids may be present in abody fluid such as blood or saliva in a cell-free state in that they arenot associated with a cell. However, the cell-free nucleic acids mayhave originally been associated with a cell, such as an endometrial cellprior to entering the bloodstream or other body fluid. In contrast,nucleic acids that are solely associated with cells in the body aregenerally not considered to be “cell-free.” For example, nucleic acidsextracted from a cellular sample are generally not considered“cell-free” as the term is used herein.

The term “clinical factors” as used herein, refers to any data that amedical practitioner may consider in determining a diagnosis orprognosis of disease. Such factors include, but are not limited to, thepatient's medical history, a physical examination of the patient,complete blood count, analysis of the activity of enzymes, examinationof cells, cytogenetics, and immunophenotyping of blood cells.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids. When a nucleotideposition in both of the molecules is occupied by nucleotides normallycapable of base pairing with each other, then the nucleic acids areconsidered to be complementary to each other at this position. Thus, twonucleic acids are substantially complementary to each other when atleast about 50%, preferably at least about 60% and more preferably atleast about 80% of corresponding positions in each of the molecules areoccupied by nucleotides which normally base pair with each other (e.g.,A:T and G:C nucleotide pairs).

As used herein, the term “diagnosis” means detecting a disease ordisorder or determining the stage or degree of a disease or disorder.Usually, a diagnosis of a disease or disorder is based on the evaluationof one or more factors and/or symptoms that are indicative of thedisease. That is, a diagnosis can be made based on the presence, absenceor amount of a factor which is indicative of presence or absence of thedisease or condition. Each factor or symptom that is considered to beindicative for the diagnosis of a particular disease does not need beexclusively related to the particular disease; i.e. there may bedifferential diagnoses that can be inferred from a diagnostic factor orsymptom. Likewise, there may be instances where a factor or symptom thatis indicative of a particular disease is present in an individual thatdoes not have the particular disease. The diagnostic methods may be usedindependently, or in combination with other diagnosing and/or stagingmethods known in the medical art for a particular disease or disorder.

As used herein, the phrase “difference of the level” refers todifferences in the quantity of a particular marker, such as a nucleicacid or a protein, in a sample as compared to a control or referencelevel. For example, the quantity of a particular biomarker may bepresent at an elevated amount or at a decreased amount in samples ofpatients with a disease compared to a reference level. In someembodiments, a “difference of a level” may be a difference between thequantity of a particular biomarker present in a sample as compared to acontrol of at least about 1%, at least about 2%, at least about 3%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 50%, at least about 60%, at least about75%, at least about 80% or more. In some embodiments, a “difference of alevel” may be a statistically significant difference between thequantity of a biomarker present in a sample as compared to a control.For example, a difference may be statistically significant if themeasured level of the biomarker falls outside of about 1.0 standarddeviations, about 1.5 standard deviations, about 2.0 standarddeviations, or about 2.5 stand deviations of the mean of any control orreference group.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a sign orsymptom of the disease or disorder, the frequency with which such a signor symptom is experienced by a patient, or both, is reduced.

The term “comparator” describes a material comprising none, or a normal,low, or high level of one of more of the marker (or biomarker)expression products of one or more the markers (or biomarkers) of thedisclosure, such that the comparator may serve as a control or referencestandard against which a sample can be compared.

The terms “dysregulated” and “dysregulation” as used herein describes adecreased (down-regulated) or increased (up-regulated) level ofexpression of a miRNA present and detected in a sample obtained fromsubject as compared to the level of expression of that miRNA in acomparator sample, such as a comparator sample obtained from one or morenormal, not-at-risk subjects, or from the same subject at a differenttime point. In some instances, the level of miRNA expression is comparedwith an average value obtained from more than one not-at-riskindividuals. In other instances, the level of miRNA expression iscompared with a miRNA level assessed in a sample obtained from onenormal, not-at-risk subject.

By the phrase “determining the level of marker (or biomarker)expression” is meant an assessment of the degree of expression of amarker in a sample at the nucleic acid or protein level, usingtechnology available to the skilled artisan to detect a sufficientportion of any marker expression product.

The terms “determining,” “measuring,” “assessing,” and “assaying” areused interchangeably and include both quantitative and qualitativemeasurement, and include determining if a characteristic, trait, orfeature is present or not. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

“Differentially increased expression” or “up regulation” refers toexpression levels which are at least 10% or more, for example, 20%, 30%,40%, or 50%, 60%, 70%, 80%, 90% higher or more, and/or 1.1 fold, 1.2fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold higher or more, and any andall whole or partial increments there between than a comparator.

“Differentially decreased expression” or “down regulation” refers toexpression levels which are at least 10% or more, for example, 20%, 30%,40%, or 50%, 60%, 70%, 80%, 90% lower or less, and/or 2.0 fold, 1.8fold, 1.6 fold, 1.4 fold, 1.2 fold, 1.1 fold or less lower, and any andall whole or partial increments there between than a comparator.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 5′-ATTGCC-3′ and 5′-TATGGC-3′ share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

“Inhibitors,” “activators,” and “modulators” of the markers are used torefer to activating, inhibitory, or modulating molecules identifiedusing in vitro and in vivo assays of endometriosis biomarkers.Inhibitors are compounds that, e.g., bind to, partially or totally blockactivity, decrease, prevent, delay activation, inactivate, desensitize,or down regulate the activity or expression of endometriosis biomarkers.“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate activity ofendometriosis biomarkers, e.g., agonists Inhibitors, activators, ormodulators also include genetically modified versions of endometriosisbiomarkers, e.g., versions with altered activity, as well as naturallyoccurring and synthetic ligands, antagonists, agonists, antibodies,peptides, cyclic peptides, nucleic acids, antisense molecules,ribozymes, RNAi, microRNA, and siRNA molecules, small organic moleculesand the like. Such assays for inhibitors and activators include, e.g.,expressing endometriosis biomarkers in vitro, in cells, or cellextracts, applying putative modulator compounds, and then determiningthe functional effects on activity, as described elsewhere herein.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,method or delivery system of the disclosure in the kit for effectingalleviation of the various diseases or disorders recited herein.Optionally, or alternately, the instructional material can describe oneor more methods of alleviating the diseases or disorders in a cell or atissue of a mammal. The instructional material of the kit of thedisclosure can, for example, be affixed to a container which containsthe identified compound, composition, vector, or delivery system of thedisclosure or be shipped together with a container which contains theidentified compound, composition, vector, or delivery system.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thecompound be used cooperatively by the recipient.

As used herein, “isolated” means altered or removed from the naturalstate through the actions, directly or indirectly, of a human being. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

“Measuring” or “measurement,” or alternatively “detecting” or“detection,” means assessing the presence, absence, quantity or amount(which can be an effective amount) of either a given substance within aclinical or subject-derived sample, including the derivation ofqualitative or quantitative concentration levels of such substances, orotherwise evaluating the values or categorization of a subject'sclinical parameters.

As used herein, “microRNA” or “miRNA” describes small non-coding RNAmolecules, generally about 15 to about 50 nucleotides in length,preferably 17-23 nucleotides, which can play a role in regulating geneexpression through, for example, a process termed RNA interference(RNAi). RNAi describes a phenomenon whereby the presence of an RNAsequence that is complementary or antisense to a sequence in a targetgene messenger RNA (mRNA) results in inhibition of expression of thetarget gene. miRNAs are processed from hairpin precursors of about 70 ormore nucleotides (pre-miRNA) which are derived from primary transcripts(pri-miRNA) through sequential cleavage by RNAse III enzymes. miRBase isa comprehensive microRNA database located at www.mirbase.org,incorporated by reference herein in its entirety for all purposes.

A “mutation,” as used herein, refers to a change in nucleic acid orpolypeptide sequence relative to a reference sequence (which ispreferably a naturally-occurring normal or “wild-type” sequence), andincludes translocations, deletions, insertions, and substitutions/pointmutations. A “mutant,” as used herein, refers to either a nucleic acidor protein comprising a mutation.

“Naturally occurring” as used herein describes a composition that can befound in nature as distinct from being artificially produced. Forexample, a nucleotide sequence present in an organism, which can beisolated from a source in nature and which has not been intentionallymodified by a person, is naturally occurring.

By “nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sulfone linkages, and combinations of suchlinkages. The term nucleic acid also specifically includes nucleic acidscomposed of bases other than the five biologically occurring bases(adenine, guanine, thymine, cytosine and uracil).

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand.” Sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences.” Sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrid,anti-sense RNA, siRNA, miRNA, genomic DNA, synthetic forms, and mixedpolymers, both sense and antisense strands, and may be chemically orbiochemically modified to contain non-natural or derivatized, synthetic,or semi-synthetic nucleotide bases. Also, included within the scope ofthe disclosure are alterations of a wild type or synthetic gene,including but not limited to deletion, insertion, substitution of one ormore nucleotides, or fusion to other polynucleotide sequences.

As used herein, a “primer” for amplification is an oligonucleotide thatspecifically anneals to a target or marker nucleotide sequence. The 3′nucleotide of the primer should be identical to the target or markersequence at a corresponding nucleotide position for optimal primerextension by a polymerase. As used herein, a “forward primer” is aprimer that anneals to the anti-sense strand of double stranded DNA(dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

As used herein, the term “providing a prognosis” refers to providing aprediction of the probable course and outcome of endometriosis,including prediction of severity, duration, chances of recovery, etc.The methods can also be used to devise a suitable therapeutic plan,e.g., by indicating whether or not the condition is still at an earlystage or if the condition has advanced to a stage where aggressivetherapy would be ineffective.

A “reference level” of a biomarker means a level of the biomarker thatis indicative of a particular disease state, phenotype, or lack thereof,as well as combinations of disease states, phenotypes, or lack thereof.A “positive” reference level of a biomarker means a level that isindicative of a particular disease state or phenotype. A “negative”reference level of a biomarker means a level that is indicative of alack of a particular disease state or phenotype.

“Sample” or “biological sample” as used herein means a biologicalmaterial isolated from an individual. The biological sample may containany biological material suitable for detecting the desired biomarkers,and may comprise cellular and/or non-cellular material obtained from theindividual.

“Standard control value” as used herein refers to a predetermined amountof a particular protein or nucleic acid that is detectable in abiological sample. The standard control value is suitable for the use ofa method of the present disclosure, in order for comparing the amount ofa protein or nucleic acid of interest that is present in a biologicalsample. An established sample serving as a standard control provides anaverage amount of the protein or nucleic acid of interest in thebiological sample that is typical for an average, healthy person ofreasonably matched background, e.g., gender, age, ethnicity, and medicalhistory. A standard control value may vary depending on the protein ornucleic acid of interest and the nature of the sample (e.g., serum).

The terms “subject,” “patient,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The terms “underexpress,” “underexpression,” “underexpressed,” or“down-regulated” interchangeably refer to a protein or nucleic acid thatis transcribed or translated at a detectably lower level in a biologicalsample from a woman with endometriosis, in comparison to a biologicalsample from a woman without endometriosis. The term includesunderexpression due to transcription, post transcriptional processing,translation, post-translational processing, cellular localization (e.g.,organelle, cytoplasm, nucleus, cell surface), and RNA and proteinstability, as compared to a control. Underexpression can be detectedusing conventional techniques for detecting mRNA (i.e., Q-PCR, RT-PCR,PCR, hybridization) or proteins (i.e., ELISA, immunohistochemicaltechniques). Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or less in comparison to a control. In certain instances,underexpression is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or morelower levels of transcription or translation in comparison to a control.

The terms “overexpress,” “overexpression,” “overexpressed,” or“up-regulated” interchangeably refer to a protein or nucleic acid (RNA)that is transcribed or translated at a detectably greater level, usuallyin a biological sample from a woman with endometriosis, in comparison toa biological sample from a woman without endometriosis. The termincludes overexpression due to transcription, post transcriptionalprocessing, translation, post-translational processing, cellularlocalization (e.g., organelle, cytoplasm, nucleus, cell surface), andRNA and protein stability, as compared to a cell from a woman withoutendometriosis. Overexpression can be detected using conventionaltechniques for detecting mRNA (i.e., Q-PCR, RT-PCR, PCR, hybridization)or proteins (i.e., ELISA, immunohistochemical techniques).Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore in comparison to a cell from a woman without endometriosis. Incertain instances, overexpression is 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-,10-fold, or more higher levels of transcription or translation incomparison to a cell from a woman without endometriosis.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialproperties of the reference molecule. Changes in the sequence of anucleic acid variant may not alter the amino acid sequence of a peptideencoded by the reference nucleic acid, or may result in amino acidsubstitutions, additions, deletions, fusions and truncations. Changes inthe sequence of peptide variants are typically limited or conservative,so that the sequences of the reference peptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference peptide can differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A variant of anucleic acid or peptide can be a naturally occurring such as an allelicvariant, or can be a variant that is not known to occur naturally.Non-naturally occurring variants of nucleic acids and peptides may bemade by mutagenesis techniques or by direct synthesis.

As used herein, the terms “treat,” “ameliorate,” “treatment,” and“treating” are used interchangeably. These terms refer to an approachfor obtaining beneficial or desired results including, but are notlimited to, therapeutic benefit and/or a prophylactic benefit.Therapeutic benefit means eradication or amelioration of the underlyingdisorder being treated. Also, a therapeutic benefit is achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the patient, notwithstanding that the patient can still beafflicted with the underlying disorder. For prophylactic benefit,treatment may be administered to a patient at risk of developing aparticular disease, or to a patient reporting one or more of thephysiological symptoms of a disease, even though a diagnosis of thisdisease may not have been made.

The term “or” as used herein and throughout the disclosure, generallymeans “and/or” unless the context dictates otherwise.

As used herein, the term “circulating miRNA” refers to any miRNA in abody fluid, regardless of whether the body fluid is traditionallyconsidered to be a part of the circulatory system. For example,“circulating miRNA” would encompass miRNA present in a subject's bloodand would also encompass miRNA present in a subject's saliva, urine, orother bodily fluid.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

In one aspect, the present disclosure relates to the discovery of a linkbetween endometriosis and alterations in circulating miRNA levels. Insome embodiments, the level of circulating miRNAs, alone and incombination with conventional endometriosis serum markers, are used toimprove endometriosis detection. In exemplary embodiment, the miRNAs areselected from the group consisting of: miR-125, miR-150, miR-342,miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755,miR-3613, miR-553, and miR-4668 and any combination thereof. Inexemplary embodiments, the miRNAs are selected from the group consistingof: miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p, miR-6755-3p,miR-3613-5p, miR-553, and miR-4668-3p and any combination thereof. Insome embodiments, the miRNA is at least two of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In someembodiments, the miRNA is at least one of the following, at least two ofthe following, or all of the following: miR-125b-5p, miR-451a, andmiR-3613-5p. In some embodiments, the miRNA is at least one of thefollowing, at least two of the following, or all of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613. In some cases, themiRNA of the disclosure further comprise let-7b.

In one aspect, the methods generally provide for the detection,measuring, and comparison of a pattern of circulating miRNA in a patientsample. In other aspects, the methods generally provide for detection,measuring and comparison of a pattern of miRNA present in a sample ofbodily fluid (e.g., blood, plasma, serum, saliva, urine). In the contextof endometriosis, it is frequently difficult to have access to thediseased cells. As such, a method that detects endometriosis using arelatively non-invasive method such as a blood draw or collection ofsaliva would be very beneficial. In various embodiments, the presentmethods overcome problems of cancer diagnosis by determining the levelsof miRNAs in the plasma of patients with liver diseases. An alteration(i.e., an increase or decrease) in the level of a miRNA gene product inthe sample obtained from the subject, relative to the level of acorresponding miRNA gene product in a control sample, is indicative ofthe presence of endometriosis in the subject. In some embodiments, thelevel of at least one miRNA gene product in the test sample is greaterthan the level of the corresponding miRNA gene product in the controlsample. In another embodiment, the level of at least one miRNA geneproduct in the test sample is less than the level of the correspondingmiRNA gene product in the control sample.

Additional diagnostic markers may be combined with the circulating miRNAlevel to construct models for predicting the presence or absence orstage of a disease. For example, clinical factors of relevance to thediagnosis of endometriosis diseases, include, but are not limited to,the patient's medical history, a physical examination, and otherbiomarkers. A diagnosis of endometriosis may also be informed by apatient's symptoms, including the type of symptoms, duration of symptomsand degree of symptoms.

Generally, the methods of this disclosure find use in diagnosing or forproviding a prognosis for endometriosis by detecting the expressionlevels of biomarkers, which are differentially expressed (up- ordown-regulated) in blood or serum from a patient. These markers can beused to distinguish the stage or severity of endometriosis. Thesemarkers can also be used to provide a prognosis for the course oftreatment in a patient with endometriosis. Similarly, these markers canbe used to diagnose infertility in a patient with endometriosis or toprovide a prognosis for a fertility trial in a patient suffering fromendometriosis. The biomarkers of the present disclosure can be usedalone or in combination for the diagnosis or prognosis of endometriosis.

In some embodiments, the methods of the present disclosure find use inassigning treatment to a patient suffering from endometriosis. Bydetecting the expression levels of biomarkers found herein, theappropriate treatment can be assigned to a patient suffering fromendometriosis. These treatments can include, but are not limited to,hormone therapy, chemotherapy, immunotherapy, and surgical treatment.Similarly, the methods of the current disclosure can be used to assigntreatment to a patient with reduced fertility due to endometriosis. Inthis fashion, by determining the degree to which the patient's fertilityhas been reduced, through the detection of biomarkers found herein, theappropriate treatment can be assigned. Relevant treatments include, butare not limited to, hormone therapy, chemotherapy, immunotherapy, andsurgical treatment.

Diagnostic and prognostic kits comprising one or more markers for useare provided herein. Also provided by the disclosure are methods foridentifying compounds that are able to prevent or treat endometriosis orreduced fertility caused by endometriosis by modulating the expressionlevel or activity of markers found in any one of the identified genesubsets. Therapeutic methods are provided, wherein endometriosis orreduced fertility caused by endometriosis is treated using an agent thattargets the markers of the disclosure.

In various embodiments, the methods of the disclosure relate to methodsof assessing a subject's risk of having or developing endometriosis,methods of assessing the severity of a subject's endometriosis, methodsof diagnosing endometriosis, methods of characterizing endometriosis,and methods of stratifying a subject having endometriosis in a clinicaltrial.

In various embodiments of the compositions and methods of the disclosuredescribed herein, the miRNA associated with endometriosis is at leastone of the following miRNAs: miR-125, miR-150, miR-342, miR-145,miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755, miR-3613,miR-553, and miR-4668. Sequences of the miRNA family members arepublicly available from miRBase at (www.mirbase.org). In someembodiments, the miRNA associated with endometriosis is at least one ofthe following miRNAs, or further comprises one of the following miRNAs:let-7, let-7a, let-7b, let-7b-3p, let-7b-5p, let-7c, let-7d, let-7e,let-7f, or let-7g. In some embodiments, the miRNA associated withendometriosis is at least two of the following: miR-125b-5p, let-7b,miR-150, miR-342, and miR-3613. In some embodiments, the miRNA is atleast one of the following, at least two of the following, or all of thefollowing: miR-125b-5p, miR-451a, and miR-3613-5p. In some embodiments,the miRNA is at least one of the following, at least two of thefollowing, or all of the following: miR-125b-5p, let-7b, miR-150,miR-342, and miR-3613.

In various embodiments of the compositions and methods of the disclosuredescribed herein, the miRNA associated with endometriosis is at leastone of the following miRNAs: miR-125b-5p, miR-150-5p, miR-342-3p,miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p,miR-126-3p, miR-6755-3p, miR-3613-5p, miR-553, and miR-4668-3p.Sequences of the miRNA family members are publicly available frommiRBase at (www.mirbase.org).

In some embodiments, the biomarkers of the disclosure include one ormore of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500, miR-451,miR-18a, miR-214, miR-126, miR-6755, miR-3613, miR-553, and miR-4668. Insome embodiments, biomarkers of the disclosure useful for diagnosingendometriosis include one or more of miR-125b, miR-150, miR-342,miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755,miR-3613, miR-553, and miR-4668. In some embodiments, biomarkers of thedisclosure useful for diagnosing endometriosis include the combinationof miR-125b, miR-451 and miR-3613. In further embodiments, thebiomarkers useful for diagnosing endometriosis further include one ormore of the following: let-7, let-7a, let-7b, let-7b-3p, let-7b-5p,let-7c, let-7d, let-7e, let-7f, or let-7g. In some specific cases, thebiomarkers further comprise let-7b. In some embodiments, the biomarkersinclude at least two of the following: miR-125b-5p, let-7b, miR-150,miR-342, and miR-3613. In some embodiments, the biomarkers include atleast one of the following, at least two of the following, or all of thefollowing: miR-125b-5p, miR-451a, and miR-3613-5p. In some embodiments,the biomarkers include at least one of the following, at least two ofthe following, or all of the following: miR-125b-5p, let-7b, miR-150,miR-342, and miR-3613. In some cases, the biomarkers may further includemiR-451.

In some embodiments, the biomarkers of the disclosure include one ormore of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p, miR-6755-3p,miR-3613-5p, miR-553, and miR-4668-3p. In some embodiments, biomarkersof the disclosure useful for diagnosing endometriosis include one ormore of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p, miR-6755-3p,miR-3613-5p, miR-553, and miR-4668-3p. In a preferred embodiment,biomarkers of the disclosure useful for diagnosing endometriosis includethe combination of miR-125b-5p, miR-451a and miR-3613-5p. In someembodiments, the biomarkers useful for diagnosing endometriosis includeat least two of the following: miR-125b-5p, let-7b, miR-150, miR-342,and miR-3613. In some embodiments, the biomarkers useful for diagnosingendometriosis include at least one of the following, at least two of thefollowing, or all of the following: miR-125b-5p, miR-451a, andmiR-3613-5p. In some embodiments, the biomarkers useful for diagnosingendometriosis include at least one of the following, at least two of thefollowing, or all of the following: miR-125b-5p, let-7b, miR-150,miR-342, and miR-3613. In some cases, they may further include miR-451.In some cases, they may further include let-7b.

In some embodiments, the biomarkers of the disclosure are one or moremiRNA associated with endometriosis which are down-regulated, orexpressed at a lower than normal level. For example, it is describedherein that miR-6755, miR-3613, miR-553, and miR-4668 are down regulatedor expressed at a lower than normal level in subjects withendometriosis. Thus, in certain embodiments, the disclosure relates tocompositions and methods useful for the diagnosis, assessment, andcharacterization of endometriosis in a subject in need thereof, basedupon the detection of a decreased level of at least one of miR-6755,miR3613, miR-553, and miR-4668.

In some embodiments, the biomarkers of the disclosure are one or moremiRNA associated with endometriosis which are down-regulated, orexpressed at a lower than normal level. For example, it is describedherein that miR-6755-3p, miR-3613-5p, miR-553, and miR-4668-3p are downregulated or expressed at a lower than normal level in subjects withendometriosis. Thus, in certain embodiments, the disclosure relates tocompositions and methods useful for the diagnosis, assessment, andcharacterization of endometriosis in a subject in need thereof, basedupon the detection of a decreased level of at least one of miR-6755-3p,miR3613-5p, miR-553, and miR-4668-3p.

In some embodiments, the biomarkers of the disclosure are one or moremiRNA associated with endometriosis which are upregulated, or expressedat a higher than normal level. For example, it is described herein thatmiR-125, miR-150, miR-342, miR-145, miR-143, miR-500, miR-451, miR-18,miR-214, and miR-126 are upregulated or expressed at a higher thannormal level in subjects with endometriosis. Thus, in certainembodiments, the disclosure relates to compositions and methods usefulfor the diagnosis, assessment, and characterization of endometriosis ina subject in need thereof, based upon the detection of an increasedlevel of at least one of miR-125, miR-150, miR-342, miR-145, miR-143,miR-500, miR-451, miR-18, miR-214, and miR-126. In some cases, one ormore of the following miRNA are upregulated or expressed at a higherthan normal level in subjects with endometriosis: let-7, let-7a, let-7b,let-7b-3p, let-7b-5p, let-7c, let-7d, let-7e, let-7f, or let-7g. In someembodiments, the biomarkers of the disclosure are one or more miRNAassociated with endometriosis which are upregulated, or expressed at ahigher than normal level. For example, it is described herein thatmiR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, and miR-126-3p areupregulated or expressed at a higher than normal level in subjects withendometriosis. Thus, in certain embodiments, the disclosure relates tocompositions and methods useful for the diagnosis, assessment, andcharacterization of endometriosis in a subject in need thereof, basedupon the detection of an increased level of at least one of miR-125b-5p,miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a,miR-18a-5p, miR-214-3p, and miR-126-3p.

In some embodiments, the disclosure provides a method for detecting amarker of endometriosis. In some embodiments, the disclosure provides amethod for monitoring the levels of miRNAs in response to treatment. Insome embodiments, the disclosure provides a method for monitoring atleast one of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p,miR-6755-3p, miR-3613-5p, miR-553, and miR-4668-3p after treatment. Insome embodiments, the disclosure provides a method for monitoring thelevels of miR-125b-5p, miR-150-5p, miR-3613-5p or any combinationthereof after endometriosis treatment. In some embodiments, thedisclosure provides a method for monitoring the levels of let-7, let-7a,let-7b, let-7b-3p, let-7b-5p, let-7c, let-7d, let-7e, let-7f, or let-7goften in addition to another miRNA provided herein. In some embodiments,the monitored miRNA is at least two of the following: miR-125b-5p,let-7b, miR-150, miR-342, and miR-3613. In some embodiments, themonitored miRNA is at least one of the following, at least two of thefollowing, or all of the following: miR-125b-5p, miR-451a, andmiR-3613-5p. In some embodiments, the monitored miRNA is at least one ofthe following, at least two of the following, or all of the following:miR-125b-5p, let-7b, miR-150, miR-342, and miR-3613.

In some cases, the method may include diagnosing endometriosis bymeasuring the level of at least one of the following miRNA: let-7a,let-7b, let-7c, let-7c, let-7d, let-7e, let-7f, and let-7g. In somecases, the method may include diagnosing endometriosis by measuring thelevel of at least two of the following miRNA: let-7a, let-7b, let-7c,let-7c, let-7d, let-7e, let-7f, and let-7g. In some cases, the methodmay include diagnosing endometriosis by measuring the level of at leastthree of the following miRNA: let-7a, let-7b, let-7c, let-7c, let-7d,let-7e, let-7f, and let-7g.

In some embodiments, the disclosure provides a method for detecting atleast one, at least two, at least three, at least four, at least five,or at least ten of the miRNAs listed in Table 1. In some embodiments,the disclosure provides a method for detecting nucleic acids that haveat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 100%homology to at least one of the sequences listed in Table 1.

TABLE 1  miRNA sequences SEQ ID NO.:  Name Sequence 1 miR-18a-3pACUGCCCUAAGUGCUCCUUCUGG 2 miR-18a-5p UAAGGUGCAUCUAGUGCAGAUAG 3miR-18b-3p UGCCCUAAAUGCCCCUUCUGGC 4 miR-18b-5p UAAGGUGCAUCUAGUGCAGUUAG 5miR-125a-3p ACAGGUGAGGUUCUUGGGAGCC 6 miR-125a-5pUCCCUGAGACCCUUUAACCUGUGA 7 miR-125b-1-3p ACGGGUUAGGCUCUUGGGAGCU 8miR-125b-2-3p UCACAAGUCAGGCUCUUGGGAC 9 miR-125b-5pUCCCUGAGACCCUAACUUGUGA 10 miR-126-3p UCGUACCGUGAGUAAUAAUGCG 11miR-126-5p CAUUAUUACUUUUGGUACGCG 12 miR-143-3p UGAGAUGAAGCACUGUAGCUC 13miR-143-5p GGUGCAGUGCUGCAUCUCUGGU 14 miR-145-3p GGAUUCCUGGAAAUACUGUUCU15 miR-145-5p GUCCAGUUUUCCCAGGAAUCCCU 16 miR-150-3pCUGGUACAGGCCUGGGGGACAG 17 miR-150-5p UCUCCCAACCCUUGUACCAGUG 18miR-214-3p ACAGCAGGCACAGACAGGCAGU 19 miR-214-5p UGCCUGUCUACACUUGCUGUGC20 miR-342-3p UCUCACACAGAAAUCGCACCCGU 21 miR-342-5pAGGGGUGCUAUCUGUGAUUGA 22 miR-451a AAACCGUUACCAUUACUGAGUU 23 miR-451bUAGCAAGAGAACCAUUACCAUU 24 miR-500a-3p AUGCACCUGGGCAAGGAUUCUG 25miR-500a-5p UAAUCCUUGCUACCUGGGUGAGA 26 miR-500b-3p GCACCCAGGCAAGGAUUCUG27 miR-500b-5p AAUCCUUGCUACCUGGGU 28 miR-553 AAAACGGUGAGAUUUUGUUUU 29miR-3613-3p ACAAAAAAAAAAGCCCAACCCUUC 30 miR-3613-5pUGUUGUACUUUUUUUUUUGUUC 31 miR-4668-3p GAAAAUCCUUUUUGUUUUUCCAG 32miR-4668-5p AGGGAAAAAAAAAAGGAUUUGUC 33 miR-6755-3pUGUUGUCAUGUUUUUUCCCUAG 34 miR-6755-5p UAGGGUAGACACUGACAACGUU 35let-7b-3p CUAUACAACCUACUGCCUUCCC 36 let-7b-5p UGAGGUAGUAGGUUGUGUGGUU

Accordingly, the disclosure may provide a new and convenient platformfor detecting a marker of endometriosis, often with relatively highsensitivity. In some embodiments, the system of the disclosure providesa platform for detecting a marker of endometriosis with at least 80%sensitivity, preferably at least 90%, preferably at least 91%,preferably at least 92%, preferably at least 93%, preferably at least94%, preferably at least 95%, preferably at least 96%, preferably atleast 97%, preferably at least 98%, preferably at least 99%, preferablyat least 100%.

In some embodiments, the system of the disclosure provides a platformfor detecting a marker of endometriosis. In some embodiments, the systemof the disclosure provides a platform for detecting a marker ofendometriosis with at least 80% specificity, preferably at least 90%,preferably at least 91%, preferably at least 92%, preferably at least93%, preferably at least 94%, preferably at least 95%, preferably atleast 96%, preferably at least 97%, preferably at least 98%, preferablyat least 99%, preferably at least 100%.

In some embodiments, the disclosure provides a system for detecting amarker of endometriosis, with at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 100% sensitivity; at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100% specificity; or both at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sensitivity andspecificity. In some embodiments, the disclosure provides a system fordetecting a marker of endometriosis with at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100% accuracy.

Accordingly, the disclosure may provide new and convenient methods fordetecting one or more markers of endometriosis, often with relativelyhigh sensitivity. In some embodiments, the methods detect or diagnoseendometriosis with at least 80% sensitivity, preferably at least 90%,preferably at least 91%, preferably at least 92%, preferably at least93%, preferably at least 94%, preferably at least 95%, preferably atleast 96%, preferably at least 97%, preferably at least 98%, preferablyat least 99%, preferably at least 100%.

In some embodiments, the methods of the disclosure detect endometriosiswith at least 80% specificity, preferably at least 90%, preferably atleast 91%, preferably at least 92%, preferably at least 93%, preferablyat least 94%, preferably at least 95%, preferably at least 96%,preferably at least 97%, preferably at least 98%, preferably at least99%, preferably at least 100%. In some embodiments, the disclosureprovides methods of detecting or diagnosing endometriosis, with at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sensitivity; atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%specificity; or both at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100% sensitivity and specificity. In some embodiments, thedisclosure provides methods of diagnosing endometriosis with at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% accuracy.

Sample Preparation

Test samples of acellular body fluid or cell-containing samples may beobtained from an individual or patient. Methods of obtaining testsamples are well-known to those of skill in the art and include, but arenot limited to aspirations or drawing of blood or other fluids. Samplesmay include, but are not limited to, whole blood, serum, plasma, saliva,cerebrospinal fluid (CSF), pericardial fluid, pleural fluid, urine, andeye fluid. In some embodiments in which the test sample contains cells,the cells may be removed from the liquid portion of the sample bymethods known in the art (e.g., centrifugation) to yield acellular bodyfluid. In suitable embodiments, serum or plasma are used as theacellular body fluid sample. Plasma and serum can be prepared from wholeblood using suitable methods well-known in the art. In theseembodiments, data may be normalized by volume of acellular body fluid.

In some embodiments, test samples of saliva may be obtained from asubject. Methods of obtaining saliva samples may include, but are notlimited to forcible ejection from the subject's mouth (e.g., spitting),aspiration, removal by a swab or other collection tool, or any othermethod known in the art. As used herein, the term “saliva” does notinclude sputum, since sputum pertains to mucus or phlegm samples. Insome embodiments, the saliva may be separated into cellular andnon-cellular fractions by methods known in the art (e.g.,centrifugation). In some embodiments, nucleic acids may be extractedfrom the cellular or non-cellular fractions.

Variability in sample preparation of cell-containing samples can becorrected by normalizing the data by, for example, protein content orcell number. In certain embodiments, the sample may be normalizedrelative to the total protein content in the sample. Total proteincontent in the sample can be determined using standard procedures,including, without limitation, Bradford assay and the Lowry method. Inother embodiments, the sample may be normalized relative to cell number.

Assays

The present disclosure relates to the discovery that the expressionlevel of particular miRNAs is associated with the presence, development,progression and severity of endometriosis. In various embodiments, thedisclosure relates to a genetic screening assay of a subject todetermine the level of expression of at least one miRNA associated withendometriosis in the subject. The present disclosure provides methods ofassessing level of at least one miRNA associated with endometriosis, aswell as methods of diagnosing a subject as having, or as being at riskof developing, endometriosis based upon the level of expression of atleast one miRNA associated with endometriosis. In some embodiments, thediagnostic assays described herein are in vitro assays.

In some embodiments, the method of the disclosure is a diagnostic assayfor assessing the presence, development, progression and severity ofendometriosis in a subject in need thereof, by determining whether thelevel of at least one miRNA associated with endometriosis is decreasedin a biological sample obtained from the subject. In variousembodiments, to determine whether the level of the at least one miRNAassociated with endometriosis is decreased in a biological sampleobtained from the subject, the level of the at least one miRNA iscompared with the level of at least one comparator control, such as apositive control, a negative control, a normal control, a wild-typecontrol, a historical control, a historical norm, or the level ofanother reference molecule in the biological sample. In someembodiments, the diagnostic assay of the disclosure is an in vitroassay. In other embodiments, the diagnostic assay of the disclosure isan in vivo assay. The miRNA identified by the assay can be any miRNAthat is associated with endometriosis. In some embodiments, the miRNA isat least one of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500,miR-451, miR-18, miR-214, miR-126, miR-6755, miR-3613, miR-553, andmiR-4668. The miRNA identified by the assay can be any miRNA that isassociated with endometriosis. In some embodiments, the miRNA is atleast one of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p,miR-6755-3p, miR-3613-5p, miR-553, and miR-4668-3p. In variousembodiments of the disclosure, the at least one miRNA associated withendometriosis is at least two miRNAs, at least three miRNAs, at leastfour miRNAs, at least five miRNAs, at least six miRNAs, at least sevenmiRNAs, at least eight miRNAs. The results of the diagnostic assay canbe used alone, or in combination with other information from thesubject, or other information from the biological sample obtained fromthe subject.

In various embodiments of the assays of the disclosure, the level of theat least one miRNA associated with endometriosis is determined to bedown-regulated when the level of the at least one miRNA is decreased byat least 10%, by at least 20%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, by at least 100%, by at least 125%, by at least 150%, by atleast 175%, by at least 200%, by at least 250%, by at least 300%, by atleast 400%, by at least 500%, by at least 600%, by at least 700%, by atleast 800%, by at least 900%, by at least 1000%, by at least 1500%, byat least 2000%, or by at least 5000%, when compared with a comparatorcontrol.

In various embodiments of the assays of the disclosure, the level of theat least one miRNA associated with endometriosis is determined to beup-regulated when the level of the at least one miRNA is increased by atleast 10%, by at least 20%, by at least 30%, by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, by at least 100%, by at least 125%, by at least 150%, by atleast 175%, by at least 200%, by at least 250%, by at least 300%, by atleast 400%, by at least 500%, by at least 600%, by at least 700%, by atleast 800%, by at least 900%, by at least 1000%, by at least 1500%, byat least 2000%, or by at least 5000%, when compared with a comparatorcontrol.

In various embodiments of the assays of the disclosure, the level of theat least one miRNA associated with endometriosis is determined to bedown-regulated when the level of the at least one miRNA is decreased byat least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, atleast 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, atleast 150-fold, at least 200-fold, or at least 500-fold, when comparedwith a comparator control. In various embodiments of the assays of thedisclosure, the level of the at least one miRNA associated withendometriosis is determined to be up-regulated when the level of the atleast one miRNA is increased by at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 5-fold, at least 10-fold, at least 25-fold, atleast 50-fold, at least 100-fold, at least 150-fold, at least 200-fold,or at least 500-fold, when compared with a comparator control.

In the assay methods of the disclosure, a test biological sample from asubject is assessed for the expression level of at least one miRNAassociated with endometriosis. The test biological sample can be an invitro sample or an in vivo sample. In various embodiments, the subjectis a human subject, and may be of any race, sex and age. Representativesubjects include those who are suspected of having endometriosis, thosewho have been diagnosed with endometriosis, those whose haveendometriosis, those who have had endometriosis, those who at risk of arecurrence of endometriosis, and those who are at risk of developingendometriosis.

In some embodiments, an endometriosis associated miRNA-binding moleculeis used in vivo for the diagnosis of endometriosis. In some embodiments,the endometriosis associated miRNA-binding molecule is nucleic acid thathybridizes with an endometriosis associated miRNA of the disclosure.

In some embodiments, the test sample is a sample containing at least afragment of a nucleic acid comprising a miRNA associated withendometriosis. The term, “fragment,” as used herein, indicates that theportion of a nucleic acid (e.g., DNA, mRNA or cDNA) that is sufficientto identify it as comprising a miRNA associated with endometriosis.

In some embodiments, the test sample is prepared from a biologicalsample obtained from the subject. The biological sample can be a samplefrom any source which contains a nucleic acid comprising endometriosisassociated miRNA, such as a body fluid (e.g., blood, plasma, serum,saliva, urine, etc.), or a tissue, or an exosome, or a cell, or acombination thereof. A biological sample can be obtained by appropriatemethods, such as, by way of examples, biopsy or fluid draw. Thebiological sample can be used as the test sample; alternatively, thebiological sample can be processed to enhance access to polypeptides,nucleic acids, or copies of nucleic acids (e.g., copies of nucleic acidscomprising a miRNA associated with endometriosis), and the processedbiological sample can then be used as the test sample. For example, invarious embodiments, nucleic acid is prepared from a biological sample,for use in the methods. Alternatively, or in addition, if desired, anamplification method can be used to amplify nucleic acids comprising allor a fragment of a nucleic acid in a biological sample, for use as thetest sample in the assessment of the expression level of a miRNAassociated with endometriosis.

The test sample is assessed to determine the level of expression of atleast one miRNA associated with endometriosis present in the nucleicacid of the subject. In general, detecting a miRNA may be carried out bydetermining the presence or absence of a nucleic acid containing a miRNAof interest in the test sample.

In some embodiments, hybridization methods, such as Northern analysis,or in situ hybridizations, can be used (see Current Protocols inMolecular Biology, 2012, Ausubel, F. et al., eds., John Wiley & Sons,including all supplements). For example, the presence of a miRNAassociated with endometriosis can be indicated by hybridization to anucleic acid probe. A “nucleic acid probe,” as used herein, can be anucleic acid probe, such as a DNA probe or an RNA probe. Forrepresentative examples of use of nucleic acid probes, see, for example,U.S. Pat. Nos. 5,288,611 and 4,851,330.

To detect at least one miRNA of interest, a hybridization sample isformed by contacting the test sample with at least one nucleic acidprobe. A preferred probe for detecting miRNA is a labeled nucleic acidprobe capable of hybridizing to miRNA. The nucleic acid probe can be,for example, a full-length nucleic acid molecule, or a portion thereof,such as an oligonucleotide of at least 10, 15, or 25 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to appropriate miRNA. The hybridization sample is maintainedunder conditions which are sufficient to allow specific hybridization ofthe nucleic acid probe to a miRNA target of interest. Specifichybridization can be performed under high stringency conditions ormoderate stringency conditions, as appropriate. In a preferredembodiment, the hybridization conditions for specific hybridization arehigh stringency. Specific hybridization, if present, is then detectedusing standard methods. If specific hybridization occurs between thenucleic acid probe and a miRNA in the test sample, the sequence that ispresent in the nucleic acid probe is also present in the miRNA of thesubject. More than one nucleic acid probe can also be used concurrentlyin this method. Specific hybridization of any one of the nucleic acidprobes is indicative of the presence of the miRNA of interest, asdescribed herein.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the hybridization methods described herein. PNAis a DNA mimic having a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1). The PNAprobe can be designed to specifically hybridize to a nucleic acidsequence comprising at least one miRNA of interest. Hybridization of thePNA probe to a nucleic acid sequence is indicative of the presence of amiRNA of interest.

Direct sequence analysis can also be used to detect miRNAs of interest.A sample comprising nucleic acid can be used, and PCR or otherappropriate methods can be used to amplify all or a fragment of thenucleic acid, and/or its flanking sequences, if desired.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequences from a subject can beused to detect, identify and quantify miRNAs associated withendometriosis. For example, in some embodiments, an oligonucleotidearray can be used. Oligonucleotide arrays typically comprise a pluralityof different oligonucleotide probes that are coupled to a surface of asubstrate in different known locations. These oligonucleotide arrays,also known as “Genechips,” have been generally described in the art, forexample, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO90/15070 and 92/10092. These arrays can generally be produced usingmechanical synthesis methods or light directed synthesis methods whichincorporate a combination of photolithographic methods and solid phaseoligonucleotide synthesis methods. See Fodor et al., Science,251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication No.WO 92/10092 and U.S. Pat. No. 5,424,186. Techniques for the synthesis ofthese arrays using mechanical synthesis methods are described in, e.g.,U.S. Pat. No. 5,384,261.

After an oligonucleotide array is prepared, a sample containing miRNA ishybridized with the array and scanned for miRNAs. Hybridization andscanning are generally carried out by methods described herein and alsoin, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995,and U.S. Pat. No. 5,424,186, the entire teachings of which areincorporated by reference herein.

In brief, a target miRNA sequence is amplified by well-knownamplification techniques, e.g., RT, PCR. Typically, this involves theuse of primer sequences that are complementary to the target miRNA.Amplified target, generally incorporating a label, is then hybridizedwith the array under appropriate conditions. Upon completion ofhybridization and washing of the array, the array is scanned todetermine the position on the array to which the target sequencehybridizes. The hybridization data obtained from the scan is typicallyin the form of fluorescence intensities as a function of location on thearray.

Other methods of nucleic acid analysis can be used to detect miRNAs ofinterest. Representative methods include direct manual sequencing (1988,Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995; 1977,Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467; Beavis et al. U.S.Pat. No. 5,288,644); automated fluorescent sequencing; single-strandedconformation polymorphism assays (SSCP); clamped denaturing gelelectrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE)(Sheffield et al., 1981, Proc. Natl. Acad. Sci. USA 86:232-236),mobility shift analysis (Orita et al., 1989, Proc. Natl. Acad. Sci. USA86:2766-2770; Rosenbaum and Reissner, 1987, Biophys. Chem. 265:1275;1991, Keen et al., Trends Genet. 7:5); RNase protection assays (Myers,et al., 1985, Science 230:1242); Luminex xMAP™ technology;high-throughput sequencing (HTS) (Gundry and Vijg, 2011, Mutat Res,doi:10.1016/j.mrfmmm.2011.10.001); next-generation sequencing (NGS)(Voelkerding et al., 2009, Clinical Chemistry 55:641-658; Su et al.,2011, Expert Rev Mol Diagn. 11:333-343; Ji and Myllykangas, 2011,Biotechnol Genet Eng Rev 27:135-158); and/or ion semiconductorsequencing (Rusk, 2011, Nature Methods doi:10.1038/nmeth.f.330; Rothberget al., 2011, Nature 475:348-352). These and other methods, alone or incombination, can be used to detect and quantity of at least one miRNA ofinterest, in a biological sample obtained from a subject. In someembodiments of the disclosure, the methods of assessing a biologicalsample to detect and quantify a miRNA of interest, as described herein,are used to diagnose, assess and characterize endometriosis in a subjectin need thereof.

In some embodiments, sequencing can be performed using a next generationsequencing assay. As used herein, the term “next generation” iswell-understood in the art and generally refers to any high-throughputsequencing approach including, but not limited to one or more of thefollowing: massively-parallel signature sequencing, pyrosequencing(e.g., using a Roche 454 sequencing device), Illumina (Solexa)sequencing, sequencing by synthesis (Illumina), Ion torrent sequencing,sequencing by ligation (e.g., SOLiD sequencing), single moleculereal-time (SMRT) sequencing (e.g., Pacific Bioscience), polonysequencing, DNA nanoball sequencing, heliscope single moleculesequencing (Helicos Biosciences), and nanopore sequencing (e.g., OxfordNanopore). In some cases, the sequencing assay uses nanopore sequencing.In some cases, the sequencing assay includes some form of Sangersequencing. In some cases, the sequencing involves shotgun sequencing;in some cases, the sequencing includes bridge PCR. In some cases, thesequencing is broad spectrum. In some cases, the sequencing is targeted.

The probes and primers according to the disclosure can be labeleddirectly or indirectly with a radioactive or nonradioactive compound, bymethods well known to those skilled in the art, in order to obtain adetectable and/or quantifiable signal; the labeling of the primers or ofthe probes according to the disclosure is carried out with radioactiveelements or with nonradioactive molecules. Among the radioactiveisotopes used, mention may be made of ³²P, ³³P, ³⁵S or ³H. Thenonradioactive entities are selected from ligands such as biotin,avidin, streptavidin or digoxigenin, haptenes, dyes, and luminescentagents such as radioluminescent, chemoluminescent, bioluminescent,fluorescent or phosphorescent agents.

Nucleic acids can be obtained from the biological sample using knowntechniques. Nucleic acid herein includes RNA, including mRNA, miRNA,etc. The nucleic acid can be double-stranded or single-stranded (i.e., asense or an antisense single strand) and can be complementary to anucleic acid encoding a polypeptide. The nucleic acid content may alsobe obtained from an extraction performed on a fresh or fixed biologicalsample.

There are many methods known in the art for the detection of specificnucleic acid sequences and new methods are continually reported. A greatmajority of the known specific nucleic acid detection methods utilizenucleic acid probes in specific hybridization reactions.

In the Northern blot, the nucleic acid probe is preferably labeled witha tag. That tag can be a radioactive isotope, a fluorescent dye or theother well-known materials. Another type of process for the specificdetection of nucleic acids of exogenous organisms in a body sample knownin the art are the hybridization methods as exemplified by U.S. Pat.Nos. 6,159,693 and 6,270,974, and related patents. To briefly summarizeone of those methods, a nucleic acid probe of at least 10 nucleotides,preferably at least 15 nucleotides, more preferably at least 25nucleotides, having a sequence complementary to a desired region of thetarget nucleic acid of interest is hybridized in a sample, subjected todepolymerizing conditions, and the sample is treated with anATP/luciferase system, which will luminesce if the nucleic sequence ispresent. In quantitative Northern blotting, levels of the polymorphicnucleic acid can be compared to wild-type levels of the nucleic acid.

A further process for the detection of hybridized nucleic acid takesadvantage of the polymerase chain reaction (PCR). The PCR process iswell known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). To briefly summarize PCR, nucleic acid primers,complementary to opposite strands of a nucleic acid amplification targetnucleic acid sequence, are permitted to anneal to the denatured sample.A DNA polymerase (typically heat stable) extends the DNA duplex from thehybridized primer. The process is repeated to amplify the nucleic acidtarget. If the nucleic acid primers do not hybridize to the sample, thenthere is no corresponding amplified PCR product.

In PCR, the nucleic acid probe can be labeled with a tag as discussedbefore. Most preferably the detection of the duplex is done using atleast one primer directed to the target nucleic acid. In yet anotherembodiment of PCR, the detection of the hybridized duplex compriseselectrophoretic gel separation followed by dye-based visualization.

Nucleic acid amplification procedures by PCR are well known and aredescribed in U.S. Pat. No. 4,683,202. Briefly, the primers anneal to thetarget nucleic acid at sites distinct from one another and in anopposite orientation. A primer annealed to the target sequence isextended by the enzymatic action of a heat stable polymerase. Theextension product is then denatured from the target sequence by heating,and the process is repeated. Successive cycling of this procedure onboth strands provides exponential amplification of the region flanked bythe primers.

Amplification is then performed using a PCR-type technique, that is tosay the PCR technique or any other related technique. Two primers,complementary to the target nucleic acid sequence are then added to thenucleic acid content along with a polymerase, and the polymeraseamplifies the DNA region between the primers.

Amplification may refer to any method for increasing the number ofcopies of a nucleic acid sequence. For example, the amplification may beperformed with a polymerase, e.g., in one or more polymerase chainreactions. Amplification may be performed using methods known in theart. These methods often depend on the product catalyzed formation ofmultiple copies of a nucleic acid or its complement. One of such methodsis polymerase chain reaction (PCR), including AFLP (amplified fragmentlength polymorphism) PCR, allele-specific PCR, Alu PCR, assembly,asymmetric PCR, colony PCR, helicase dependent PCR, hot start PCR,inverse PCR, in situ PCR, intersequence-specific PCR or IS SR PCR,digital PCR, droplet digital PCR, linear-after-the-exponential-PCR orLate PCR, long PCR, nested PCR, real-time PCR, duplex PCR, multiplexPCR, quantitative PCR, or single cell PCR. Other amplification methodsmay also be used, including ligase chain reaction (LCR), nucleic acidsequence based amplification (NASBA), linear amplification, isothermallinear amplification, Q-beta-replicase method, 3SR, TranscriptionMediated Amplification (TMA), Strand Displacement Amplification (SDA),or Rolling Circle Amplification (RCA).

Stem-loop RT-PCR is a PCR method that is useful in the methods of thedisclosure to amplify and quantify miRNAs of interest (See Caifu et al.,2005, Nucleic Acids Research 33:e179; Mestdagh et al., 2008, NucleicAcids Research 36:e143; Varkonyi-Gasic et al., 2011, Methods Mol Biol.744:145-57). Briefly, the method includes two steps: RT and real-timePCR. First, a stem-loop RT primer is hybridized to a miRNA molecule andthen reverse transcribed with a reverse transcriptase. Then, the RTproducts are quantified using conventional real-time PCR.

The expression specifically hybridizing in stringent conditions refersto a hybridizing step in the process of the disclosure where theoligonucleotide sequences selected as probes or primers are of adequatelength and sufficiently unambiguous so as to minimize the amount ofnon-specific binding that may occur during the amplification. Theoligonucleotide probes or primers herein described may be prepared byany suitable methods such as chemical synthesis methods.

Hybridization is typically accomplished by annealing the oligonucleotideprobe or primer to the template nucleic acid under conditions ofstringency that prevent non-specific binding but permit binding of thistemplate nucleic acid which has a significant level of homology with theprobe or primer.

Among the conditions of stringency is the melting temperature (Tm) forthe amplification step using the set of primers, which is in the rangeof about 50° C. to about 95° C. Typical hybridization and washingstringency conditions depend in part on the size (i.e., number ofnucleotides in length) of the template nucleic acid or theoligonucleotide probe, the base composition and monovalent and divalentcation concentrations (Ausubel et al., 1994, eds Current Protocols inMolecular Biology).

In a preferred embodiment, the process for determining the quantitativeand qualitative profile according to the present disclosure ischaracterized in that the amplifications are real-time amplificationsperformed using a labeled probe, preferably a labeled hydrolysis-probe,capable of specifically hybridizing in stringent conditions with asegment of a nucleic acid sequence, or polymorphic nucleic acidsequence. The labeled probe is capable of emitting a detectable signalevery time each amplification cycle occurs.

The real-time amplification, such as real-time PCR, is well known in theart, and the various known techniques will be employed in the best wayfor the implementation of the present process. These techniques areperformed using various categories of probes, such as hydrolysis probes,hybridization adjacent probes, or molecular beacons. The techniquesemploying hydrolysis probes or molecular beacons are based on the use ofa fluorescence quencher/reporter system, and the hybridization adjacentprobes are based on the use of fluorescence acceptor/donor molecules.

Hydrolysis probes with a fluorescence quencher/reporter system areavailable in the market, and are for example commercialized by theApplied Biosystems group (USA). Many fluorescent dyes may be employed,such as FAM dyes (6-carboxy-fluorescein), or any other dyephosphoramidite reagents.

Among the stringent conditions applied for any one of thehydrolysis-probes of the present disclosure is the Tm, which is in therange of about 50° C. to 95° C. Preferably, the Tm for any one of thehydrolysis-probes of the present disclosure is in the range of about 55°C. to about 80° C. Most preferably, the Tm applied for any one of thehydrolysis-probes of the present disclosure is about 75° C.

In another preferred embodiment, the process for determining thequantitative and qualitative profile according to the present disclosureis characterized in that the amplification products can be elongated,wherein the elongation products are separated relative to their length.The signal obtained for the elongation products is measured, and thequantitative and qualitative profile of the labeling intensity relativeto the elongation product length is established.

The elongation step, also called a run-off reaction, allows one todetermine the length of the amplification product. The length can bedetermined using conventional techniques, for example, using gels suchas polyacrylamide gels for the separation, DNA sequencers, and adaptedsoftware. Because some mutations display length heterogeneity, somemutations can be determined by a change in length of elongationproducts.

In one aspect, the disclosure includes a primer that is complementary toa nucleic acid sequence of the miRNA of interest, and more particularlythe primer includes 12 or more contiguous nucleotides substantiallycomplementary to the sequence of the miRNA of interest. Preferably, aprimer featured in the disclosure includes a nucleotide sequencesufficiently complementary to hybridize to a nucleic acid sequence ofabout 12 to 25 nucleotides. More preferably, the primer differs by nomore than 1, 2, or 3 nucleotides from the target nucleotide sequence. Inanother aspect, the length of the primer can vary in length, preferablyabout 15 to 28 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27 or 28 nucleotides in length).

Determining Effectiveness of Therapy or Prognosis

In one aspect, the level of one or more circulating miRNAs in abiological sample of a patient is used to monitor the effectiveness oftreatment or the prognosis of disease. In some embodiments, the level ofone or more circulating miRNAs in a test sample obtained from a treatedpatient can be compared to the level from a reference sample obtainedfrom that patient prior to initiation of a treatment. Clinicalmonitoring of treatment typically entails that each patient serve as hisor her own baseline control. In some embodiments, test samples areobtained at multiple time points following administration of thetreatment. In these embodiments, measurement of level of one or morecirculating miRNAs in the test samples provides an indication of theextent and duration of in vivo effect of the treatment.

Measurement of biomarker levels allow for the course of treatment of adisease to be monitored. The effectiveness of a treatment regimen for adisease can be monitored by detecting one or more biomarkers in aneffective amount from samples obtained from a subject over time andcomparing the amount of biomarkers detected. For example, a first samplecan be obtained prior to the subject receiving treatment and one or moresubsequent samples are taken after or during treatment of the subject.Changes in biomarker levels across the samples may provide an indicationas to the effectiveness of the therapy.

In some embodiments, the disclosure provides a method for monitoring thelevels of miRNAs in response to treatment. For example, in certainembodiments, the disclosure provides for a method of determining theefficacy of treatment in a subject, by measuring the levels of one ormore miRNAs described herein. In some embodiments, the level of the oneor more miRNAs can be measured over time, where the level at onetimepoint after the initiation of treatment is compared to the level atanother timepoint after the initiation of treatment. In someembodiments, the level of the one or more miRNAs can be measured overtime, where the level at one timepoint after the initiation of treatmentis compared to the level prior to the initiation of treatment. In someembodiments, the disclosure provides a method for monitoring at leastone of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500, miR-451,miR-18, miR-214, miR-126, miR-6755, miR-3613, miR-553, and miR-4668after treatment. In some embodiments, the disclosure provides a methodfor monitoring the levels of miR-125, miR-150, miR-3613 or anycombination thereof after endometriosis treatment.

In some embodiments, the disclosure provides a method for monitoring atleast one of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, miR-126-3p,miR-6755-3p, miR-3613-5p, miR-553, and miR-4668-3p after treatment. Insome embodiments, the disclosure provides a method for monitoring thelevels of miR-125, miR-150, miR3613 or any combination thereof afterendometriosis treatment.

In some embodiments, the disclosure provides a method for assessing theefficacy of an endometriosis treatment. For example, in someembodiments, the method indicates that the treatment is effective whenthe level of miR-125 is decreased in a sample of a treated subject ascompared to a control diseased subject or population not receivingtreatment. In some embodiments, the method indicates that the treatmentis effective when the level of miR-150 is increased in a sample of atreated subject as compared to a control diseased subject or populationnot receiving treatment. In some embodiments, the method indicates thatthe treatment is effective when the level of miR-3613 is increased in asample of a treated subject as compared to a control diseased subject orpopulation not receiving treatment.

In some embodiments, the disclosure provides a method for assessing theefficacy of an endometriosis treatment. For example, in someembodiments, the method indicates that the treatment is effective whenthe level of miR-125b-5p is decreased in a sample of a treated subjectas compared to a control diseased subject or population not receivingtreatment. In some embodiments, the method indicates that the treatmentis effective when the level of miR-150-5p is increased in a sample of atreated subject as compared to a control diseased subject or populationnot receiving treatment. In some embodiments, the method indicates thatthe treatment is effective when the level of miR-3613-5p is increased ina sample of a treated subject as compared to a control diseased subjector population not receiving treatment.

To identify therapeutics or drugs that are appropriate for a specificsubject, a test sample from the subject can also be exposed to atherapeutic agent or a drug, and the level of one or more biomarkers canbe determined. Biomarker levels can be compared to a sample derived fromthe subject before and after treatment or exposure to a therapeuticagent or a drug, or can be compared to samples derived from one or moresubjects who have shown improvements relative to a disease as a resultof such treatment or exposure. Thus, in one aspect, the disclosureprovides a method of assessing the efficacy of a therapy with respect toa subject comprising taking a first measurement of a biomarker panel ina first sample from the subject; effecting the therapy with respect tothe subject; taking a second measurement of the biomarker panel in asecond sample from the subject and comparing the first and secondmeasurements to assess the efficacy of the therapy.

Additionally, therapeutic agents suitable for administration to aparticular subject can be identified by detecting one or more biomarkersin an effective amount from a sample obtained from a subject andexposing the subject-derived sample to a test compound that determinesthe amount of the biomarker(s) in the subject-derived sample.Accordingly, treatments or therapeutic regimens for use in subjectshaving endometriosis can be selected based on the amounts of biomarkersin samples obtained from the subjects and compared to a reference value.Two or more treatments or therapeutic regimens can be evaluated inparallel to determine which treatment or therapeutic regimen would bethe most efficacious for use in a subject to delay onset, or slowprogression of a disease. In various embodiments, a recommendation ismade on whether to initiate or continue treatment of a disease.

A prognosis may be expressed as the amount of time a patient can beexpected to survive. Alternatively, a prognosis may refer to thelikelihood that the disease goes into remission or to the amount of timethe disease can be expected to remain in remission. Prognosis can beexpressed in various ways; for example, prognosis can be expressed as apercent chance that a patient will survive after one year, five years,ten years or the like. Alternatively, prognosis may be expressed as thenumber of years, on average that a patient can expect to survive as aresult of a condition or disease. The prognosis of a patient may beconsidered as an expression of relativism, with many factors affectingthe ultimate outcome. For example, for patients with certain conditions,prognosis can be appropriately expressed as the likelihood that acondition may be treatable or curable, or the likelihood that a diseasewill go into remission, whereas for patients with more severe conditionsprognosis may be more appropriately expressed as likelihood of survivalfor a specified period of time. Additionally, a change in a clinicalfactor from a baseline level may impact a patient's prognosis, and thedegree of change in level of the clinical factor may be related to theseverity of adverse events. Statistical significance is often determinedby comparing two or more populations, and determining a confidenceinterval and/or a p value.

Multiple determinations of circulating miRNA levels can be made, and atemporal change in activity can be used to determine a prognosis. Forexample, comparative measurements are made of the circulating miRNA ofan acellular body fluid in a patient at multiple time points, and acomparison of a circulating miRNA value at two or more time points maybe indicative of a particular prognosis.

In certain embodiments, the levels of activity of one or morecirculating miRNAs are used as indicators of an unfavorable prognosis.According to the method, the determination of prognosis can be performedby comparing the measured circulating miRNA level to levels determinedin comparable samples from healthy individuals or to levels known tocorresponding with favorable or unfavorable outcomes. The circulatingmiRNA levels obtained may depend on a number of factors, including, butnot limited to, the laboratory performing the assays, the assay methodsused, the type of body fluid sample used and the type of disease apatient is afflicted with. According to the method, values can becollected from a series of patients with a particular disorder todetermine appropriate reference ranges of circulating miRNA for thatdisorder. One of ordinary skill in the art is capable of performing aretrospective study that compares the determined levels to the observedoutcome of the patients and establishing ranges of levels that can beused to designate the prognosis of the patients with a particulardisorder. For example, levels in the lowest range would be indicative ofa more favorable prognosis, while circulating miRNA levels in thehighest ranges would be indicative of an unfavorable prognosis. Thus, inthis aspect the term “elevated levels” refers to levels of that areabove the range of the reference value. In some embodiments patientswith “high” or “elevated” levels have levels that are higher than themedian activity in a population of patients with that disease. Incertain embodiments, “high” or “elevated” levels for a patient with aparticular disease refers to levels that are above the median values forpatients with that disorder and are in the upper 40% of patients withthe disorder, or to levels that are in the upper 20% of patients withthe disorder, or to levels that are in the upper 10% of patients withthe disorder, or to levels that are in the upper 5% of patients with thedisorder.

Because the level of circulating miRNA in a test sample from a patientrelates to the prognosis of a patient in a continuous fashion, thedetermination of prognosis can be performed using statistical analysesto relate the determined circulating miRNA levels to the prognosis ofthe patient. A skilled artisan is capable of designing appropriatestatistical methods. For example, the methods may employ the chi-squaredtest, the Kaplan-Meier method, the log-rank test, multivariate logisticregression analysis, Cox's proportional-hazard model and the like indetermining the prognosis. Computers and computer software programs maybe used in organizing data and performing statistical analyses. Theapproach by Giles et. al., British Journal of Hemotology, 121:578-585,is exemplary. As in Giles et al., associations between categoricalvariables (e.g., miRNA levels and clinical characteristics) can beassessed via cross-tabulation and Fisher's exact test. Unadjustedsurvival probabilities can be estimated using the method of Kaplan andMeier. The Cox proportional hazards regression model also can be used toassess the ability of patient characteristics (such as miRNA levels) topredict survival, with ‘goodness of fit’ assessed by theGrambsch-Therneau test, Schoenfeld residual plots, martingale residualplots and likelihood ratio statistics (see Grambsch et al, 1995). Insome embodiments, this approach can be adapted as a simple computerprogram that can be used with personal computers or personal digitalassistants (PDA). The prediction of patients' survival time in based ontheir circulating miRNA levels can be performed via the use of a visualbasic for applications (VBA) computer program developed within MicrosoftExcel. The core construction and analysis may be based on the Coxproportional hazard models. The VBA application can be developed byobtaining a base hazard rate and parameter estimates. These statisticalanalyses can be performed using a statistical program such as the SASproportional hazards regression, PHREG, procedure. Estimates can then beused to obtain probabilities of surviving from one to 24 months giventhe patient's covariates. The program can make use of estimatedprobabilities to create a graphical representation of a given patient'spredicted survival curve. In certain embodiments, the program alsoprovides 6-month, 1-year and 18-month survival probabilities. Agraphical interface can be used to input patient characteristics in auser-friendly manner. In some embodiments of the disclosure, multipleprognostic factors, including circulating miRNA level, are consideredwhen determining the prognosis of a patient. For example, the prognosisof an endometriosis subject or may be determined based on the presenceof miRNA in a body fluid and one or more prognostic factors selectedfrom the group consisting of cytogenetics, performance status, age,gender and previous diagnosis. In another example, the prognosis of acancer patient may be determined based on circulating miRNA and one ormore prognostic factors selected from the group consisting ofcytogenetics, performance status, age, gender and previous diagnosis. Incertain embodiments, other prognostic factors may be combined with thecirculating miRNA level or other biomarkers in the algorithm todetermine prognosis with greater accuracy.

Treatment

The present disclosure provides therapeutic molecules for the treatmentor prevention of endometriosis. In some embodiments, the therapeuticmolecules include but are not limited to inhibitors, activators, andmodulators of the markers of the disclosure. For example, if a gene isdownregulated in endometriosis, than it would be desirable to increasethe expression of the downregulated gene to normal levels using anactivator as a form of therapy. Activators are compounds that increase,open, activate, facilitate, enhance activation, sensitize, agonize, orup regulate activity of endometriosis biomarkers. Alternatively, if agene is upregulated in endometriosis, than it would be desirable todecrease the expression of the upregulated gene to normal levels usingan inhibitor as a form of therapy. Inhibitors are compounds that, e.g.,bind to, partially or totally block activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity orexpression of endometriosis biomarkers.

Methods and materials for increasing or decreasing the expression levelsof the markers of the present disclosure are well known and within theskill of a person in the art. A non-limitative list of known methods andmaterials includes: diet, vitamins, dietary supplements, gene therapymethods, antisense oligonucleotides, drugs and hormonal medications.

The disclosure provides a method of treating endometriosis by targetingthe miRNAs described herein. For example, in some embodiments, thedisclosure provides a method of treating endometriosis in a subjectcomprising administering an agent that increases the expression or levelof one more miRNAs described herein. For example, in some embodiments,the method comprises administering an agent that increases theexpression or level of one more of miR-6755, miR3613, miR-553, andmiR-4668. In some embodiments, the disclosure provides a method oftreating endometriosis in a subject comprising administering an agentthat decreases the expression or level of one more miRNAs describedherein. For example, in some embodiments, the method comprisesadministering an agent that decreases the expression or level of onemore of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500, miR-451,miR-18, miR-214, and miR-126.

The disclosure provides a method of treating endometriosis by targetingthe miRNAs described herein. For example, in some embodiments, thedisclosure provides a method of treating endometriosis in a subjectcomprising administering an agent that increases the expression or levelof one more miRNAs described herein. For example, in some embodiments,the method comprises administering an agent that increases theexpression or level of one more of miR-6755-3p, miR3613-5p, miR-553, andmiR-4668-3p. In some embodiments, the disclosure provides a method oftreating endometriosis in a subject comprising administering an agentthat decreases the expression or level of one more miRNAs describedherein. For example, in some embodiments, the method comprisesadministering an agent that decreases the expression or level of onemore of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p,miR-500a-3p, miR-451a, miR-18a-5p, miR-214-3p, and miR-126-3p.

Once a patient is diagnosed with having or is at risk of havingendometriosis, the patient can be treated using methods known in theart. Well known treatments for endometriosis include, but are notlimited to, pain killers, hormonal treatments, chemotherapy, andsurgical treatments. Pain killers used for the treatment ofendometriosis include both simple analgesics, such as paracetamol, COX-2inhibitors, aspirin, and other non-steroidal anti-inflammatory drugswell known in the art, and narcotic analgesics, such as morphine,codine, oxycodone, and others well known in the art. Hormonal treatmentsinclude, but are not limited to, oral contraceptives, progestins, suchas Dydrogesterone, Medroxyprogesterone acetate, Depotmedroxyprogesterone acetate, Norethisterone, Levonorgestrel, and otherswell known in the art, progesterone and progesterone-like substances,GnRH agonists, such as leuprorelin, buserelin, goserelin, histrelin,deslorelin, nafarelin, and triptorelin, androgens and syntheticandrogens like Danazol, and aromatase inhibitors. Surgical treatmentsinclude, but are not limited to, laparoscopic surgery, hysterectomy, andoophorectomy. Other treatments particularly well suited for use in thepresent disclosure are well known in the art. In some embodiments, thepatient can be treated using a statin, including but not limited to,atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin and simvastatin.

Kits

The present disclosure also pertains to kits useful in the methods ofthe disclosure. Such kits comprise components useful in any of themethods described herein, including for example, hybridization probes orprimers (e.g., labeled probes or primers), reagents for detection oflabeled molecules, oligonucleotide arrays, restriction enzymes,antibodies, allele-specific oligonucleotides, means for amplification ofa subject's nucleic acids, means for reverse transcribing a subject'sRNA, means for analyzing a subject's nucleic acid sequence, andinstructional materials. For example, in some embodiments, the kitcomprises components useful for the detection and quantification of atleast one miRNA associated with endometriosis. In a preferred embodimentof the disclosure, the kit comprises components for detecting one ormore of the miRNAs associated with endometriosis as elsewhere describedherein.

The present disclosure also provides kits for diagnosing endometriosisor reduced fertility caused by endometriosis, comprising a probe for oneor more nucleic acid biomarkers known to be differentially expressed inendometriosis. In one particular embodiment, the kit comprises reagentsfor quantitative amplification of the selected biomarkers.Alternatively, the kit may comprise a microarray. In some embodimentsthe kit comprises 2 or more probes. In other embodiments, the kits maycontain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more probes.

The present disclosure also pertains to kits useful in the methods ofthe disclosure. Such kits comprise various combinations of componentsuseful in any of the methods described elsewhere herein, including forexample, materials for quantitatively analyzing a biomarker of thedisclosure (e.g., polypeptide and/or nucleic acid), materials forassessing the activity of a biomarker of the disclosure (e.g.,polypeptide and/or nucleic acid), and instructional material. Forexample, in some embodiments, the kit comprises components useful forthe quantification of a desired nucleic acid in a biological sample.

In a further embodiment, the kit comprises the components of an assayfor monitoring the effectiveness of a treatment administered to asubject in need thereof, containing instructional material and thecomponents for determining whether the level of a biomarker of thedisclosure in a biological sample obtained from the subject is modulatedduring or after administration of the treatment. In various embodiments,to determine whether the level of a biomarker of the disclosure ismodulated in a biological sample obtained from the subject, the level ofthe biomarker is compared with the level of at least one comparatorcontrol contained in the kit, such as a positive control, a negativecontrol, a historical control, a historical norm, or the level ofanother reference molecule in the biological sample. In certainembodiments, the ratio of the biomarker and a reference molecule isdetermined to aid in the monitoring of the treatment.

EXPERIMENTAL EXAMPLES

The disclosure is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the disclosure should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present disclosure andpractice the claimed methods. The following working examples therefore,specifically point out the preferred embodiments of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: Serum Micro RNAs as Diagnostic Markers of Endometriosis; aComprehensive Array Based Analysis

Microarray studies from eutopic and ectopic endometrial tissue of womenwith and without endometriosis show differential expression of severalmiRNAs (Ohlsson et al., 2009, Mol Endocrinol, 23:265-275, Petracco etal., 2011, J Clin Endocrinol Metab, 96:E1925-1933). They play animportant role in the pathogenesis of endometriosis and relatedinfertility by regulating gene expression (Teague et al., 2010, HumReprod Update, 16:142-165). The tissue miRNAs are shed from thepathologic tissues to the circulation, and a strong correlation has beenshown between circulating and tissue levels (Resnick et al., 2009,Gynecol Oncol, 112:55-59). Several differentially expressed miRNA havebeen identified in serum of endometriosis patients (Jia et al., 2013,Hum Reprod, 28:322-330, Wang et al., 2013, J Clin Endocrinol Metab,98:281-289). Previously it has been demonstrated that let-7b andmiR-135a were differentially expressed in the serum from endometriosispatients and are useful biomarkers of the disease (Cho et al., 2015,Fertil Steril, 103(5):1252-1260). In this study, it has beendemonstrated that other miRNAs are markers of this disease. This study,is a comprehensive evaluation of the global miRNA profile in serum ofwomen with endometriosis and identifying several miRNAs used asnoninvasive biomarkers of the disease.

The materials and methods employed in these experiments are nowdescribed.

Study Population

Serum samples were collected from 48 women with and withoutendometriosis. The study population was selected from patients thatunderwent laparascopy for multiple benign indications including pelvicmasses, pelvic pain, infertility, and endometriosis between June 2010and March 2013. Patients gave informed consent to participate in thestudy. Criteria for inclusion were age 20 to 50 years, no hormonaltherapy for at least 3 months preceding surgery, no other inflammatorydisease. The exclusion criteria included postmenopausal status, previoushormone or gonadotropin-releasing hormone (GnRH) agonist use within 3months of the surgery, adenomyosis, endometrial cancer, hyperplasia, orendometrial polyps, infectious diseases, chronic or acute inflammatorydiseases, malignancy, autoimmune disease, and cardiovascular disease.Pretreatment serum CA-125 levels in all patients were measured usingCA-125 II electro-chemiluminescence immunoassay (ECLIA) with theRoche/Hitachi Modular Analytics E170 system (Roche Diagnostics). Duringsurgery, all possible endometriotic lesions were excised and examinedfor the final diagnosis. Patients were assigned to the endometriosisgroup after pathologic confirmation of the excised tissue. The extent ofendometriosis was determined using the American Society of ReproductiveMedicine (ASRM) revised classification (Revised American Society forReproductive Medicine classification of endometriosis. Fertil Steril1996, 67:817-821). Twenty-four patients had histologically confirmedperitoneal and/or ovarian endometriosis, with moderate-to-severe disease(stages III and IV). Twenty-four patients participated as controls,which included 10 cases of dermoid cysts (n=10), serous cystadenoma(n=5), mucinous cystadenoma (n=3), simple ovarian cysts (n=5), andparatubal cysts (n=1).

Sample Collection and RNA Extraction

Blood samples (10 ml) were collected after 8 hours of fasting beforesurgery. Sterile tubes containing no additives were used. The sampleswere immediately centrifuged and the serums were frozen at −80° C. forfurther analysis. Half of the sample from each individual were pooledfor microarray analysis and the remaining half of the sample was usedfor individual analysis of micro RNAs. The total RNA was extracted from400 μl of serum using the miRVana RNA Isolation Kit (Applied Biosystems)according to the manufacturer's specifications and was eluted with 50 mlof nuclease-free water. The yield of RNA was determined using a NanoDrop ND-2000-spectrophotometer (Nano drop Technologies, USA).

miRNA Microarray Expression Profiling

Total RNAs from the pooled samples of women with endometriosis andcontrols were used for microRNA microarray profiling. Total RNA (100 ng)was labeled with the microRNA. Complete Labeling and Hyb Kit(Affymetrix, USA) and hybridized on the Human microRNA Microarray Kit(Release 16.0, Affymetrix), which contains 60000 probes for 1205 humanand 144 human viral microRNAs. Hybridization signals were detected withthe Affymetrix Microarray Scanner (Affymetrix) and the scanned imageswere analyzed using Affymetrix Feature Extraction Software (Affymetrix).

Quantitative Real-Time Polymerase Chain Reaction for miRNAs

Invitrogen NCode miRNA First-Strand cDNA Synthesis MIRC-50 kit (LifeTechnologies) following the manufacturer's instructions. Total RNA (25ng) from each sample was reverse transcribed, and the miRNAs werequantified using the iQ SYBR Green supermix kit (Bio-Rad Laboratories)with the specific forward primers for miR-125b-5p (SEQ ID NO:37),miR-150-5p (SEQ ID NO:38), miR-342-3p (SEQ ID NO:39), miR-145-5p (SEQ IDNO:40), miR-143-3p (SEQ ID NO:41), miR-500a-3p (SEQ ID NO:42), miR-451a(SEQ ID NO:43), miR-18a-5p (SEQ ID NO:44), miR-6755-3p (SEQ ID NO:45),miR-3613-5p (SEQ ID NO:46), miR-553 (SEQ ID NO:47), miR-4668-3p (SEQ IDNO:48) and the universal reverse primer complementary to the anchorprimer:

(SEQ ID NO: 37) miR-125b-5p forward UCCCUGAGACCCUAACUUGUGA;(SEQ ID NO: 38) miR-150-5p forward UCUCCCAACCCUUGUACCAGUG;(SEQ ID NO: 39) miR-342-3p forward UCUCACACAGAAAUCGCACCCGU;(SEQ ID NO: 40) miR-145-5p forward GUCCAGUUUUCCCAGGAAUCCCU;(SEQ ID NO: 41) miR-143-3p forward UGAGAUGAAGCACUGUAGCUC;(SEQ ID NO: 42) miR-500a-3p forward AUGCACCUGGGCAAGGAUUCUG;(SEQ ID NO: 43) miR-451a forward AAACCGUUACCAUUACUGAGUU; (SEQ ID NO: 44)miR-18a-5p forward UAAGGUGCAUCUAGUGCAGAUAG; (SEQ ID NO: 45)miR-6755-3p forward UGUUGUCAUGUUUUUUCCCUAG; (SEQ ID NO: 46)miR-3613-5p forward UGUUGUACUUUUUUUUUUGUUC; (SEQ ID NO: 47)miR-553 forward AAAACGGUGAGAUUUUGUUUU; (SEQ ID NO: 48)miR-4668-3p forward GAAAAUCCUUUUUGUUUUUCCAG; (SEQ ID NO: 49)miR-214-3p forward ACAGCAGGCACAGACAGGCAGU; (SEQ ID NO: 50)miR-126-3p forward UCGUACCGUGAGUAAUAAUGCG;

The reaction mixture included 4 μl of cDNA, 5 μl of iQSYBR GreenSupermix, 0.5 μl of forward primer, 0.5 μl of universal quantitativepolymerase chain reaction primer for a final reaction volume of 10 μl.The thermal cycling conditions were initiated by uracil-N-glycosylaseactivation at 50° C. for 2 minutes and initial denaturation at 95° C.for 15 minutes, followed by 50 cycles at 95° C. for 15 seconds andannealing at 59° C. for 50 seconds. Threshold cycle and melting curveswere acquired by using the quantitation and melting curve program of theBio-Rad iCycleri Qsystem (Bio-Rad Laboratories). Anchorreverse-transcription primer was used as the template for negativecontrol, and U6 (U6 forward, CTCGCTTCGGCAGCACA) (SEQ ID NO:51) smallnuclear RNA was used as a control to determine relative miRNA expression(Kuwabara et al., 2011, Circ Cardiovasc Genet, 4:446-454). The relativemRNA level was determined using comparative cycle threshold (Ct) method(known as 2^(ΔΔ)CT method).

Statistical Analysis

Student's t-test and Fisher's exact test were used determine thestatistical significance of differences in clinical characteristicsbetween the endometriosis and control groups. The expression levels ofserum miRNAs between the groups were compared using the Mann-Whitney Utest. Receiver operating characteristics (ROC) curves and the area underthe ROC curve (AUC) were established to evaluate the diagnostic value ofplasma microRNAs for differentiating between endometriosis and controls.Based on ROC analysis, the best statistical cut-off values of plasmamiRNAs was calculated, and the sensitivity and specificity for selectedcut-off points were then assessed. All statistical analysis wasperformed using SPSS 16.0 (SPSS Inc, Chicago, Ill.). P<0.05 wasconsidered statistically significant.

The results of the experiments are now described.

Clinical Characteristics

The mean (±SD) age of the women with endometriosis was 33.08±6.63 yearsand 32.16±9.46 years of controls (p>0.05). There was also nostatistically significant difference between the other clinicalparameters including gravidity, parity, alcohol usage and othercomorbidities. Pelvic pain intensity of the endometriosis groupdetermined with VAS (visual analogue scale) was higher than the controlgroup (5.4±3.4, 2.1±2.4 respectively, p<0.001). CA-125 values weresignificantly higher in endometriosis group than control group buthighly variable (103.3±121.7, 17.5±6.3 respectively, p<0.003). All womenwith endometriosis had ovarian endometriosis, 16 of them had peritonealdisease and 8 of them had deep infiltrating endometriosis.

Expression of Circulating miRNAs in Serum of Study Population

Affymetrix miRNA 4.0 Arrays were used to detect miRNA profile of thepooled samples and analyzed a total of 36,354 miRNAs from these arraystudies. Out of these miRNAs 11,653 were down regulated and 10,010 wereupregulated. Differentially expressed miRNAs with more than 10-foldchange in their expression between the endometriosis and the controlgroups were selected (FIG. 1). As shown in FIG. 1, miR-125b-5p,miR-150-5p, miR-342-3p, miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a,miR-18a-5p were upregulated while miR-3613-5p, and miR-6755-3p were downregulated.

The relative expression of miR-125b-5p, miR-150-5p, miR-342-3p,miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-6755-3p,miR-3613-5p in the serum of women with and without endometriosis wereassessed using real-time quantitative polymerase chain reaction.Expression levels of miR-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p were unregulated (p<0.05),while miR-6755-3p and miR-3613-5p levels were downregulated in womenwith endometriosis (p<0.05) as shown in FIG. 2A and FIG. 2B. Thevariation of these miRNAs through the menstrual cycle was alsodetermined. In the endometriosis group there were 8 and 16 proliferativeand secretory samples, respectively. In the control group there were 13proliferative and 11 secretory: samples. There was no differentialmenstrual cyclic stage specific expression of these miRNAs amongendometriotic patients (p>0.05).

Assessment of the Diagnostic Value of Circulating miRNAs inEndometriosis

The ROC curve analysis of serum miRNAs that were differentiallyexpressed between the two groups, was performed. AUC values of thedifferentially expressed miRNAs are shown in Table 2 and FIG. 3. Amongthe differentially expressed miRNAs, miR-125b-5p expression levels hadthe highest AUC (0.974, 95% Confidence Interval [CI], 0.00-1.00,p<0.001), with a sensitivity and specificity of 100% and 96%,respectively, for the cut off value of 0.0688. miRNA expressions indiagnosing endometriosis was improved by applying a logistic regressionmodel to data with disease versus control samples. Since thedistributions of all the biomarkers were not skewed normally, thevariables were logarithmically transformed (FIGS. 3A-3C). A high AUC wasobserved for miR-125b-5p alone. A further increase in the AUC to 1.000was achieved when combining the predictors 125b-5p, 451a and 3613-5p(FIG. 3D). The sensitivity and specificity reached 100% when these threemicroRNAs were combined. Therefore, the combination of micro RNAsdemonstrated improved diagnostic ability over any individual micro RNAs.The following logistic model was selected: miRNAcombination=118.406+108.751×log 10(125b-5p)+41.015×log10(451a)−57.935×log 10(3613-5p). The microarray data shown in Table 3demonstrates the top miRNA hits and the comparison of endometriosis (E1)upregulation vs. control (C1), E1 downregulation vs. C1.

The Use for microRNAs as Biomarkers for Endometriosis

In this study, the differentially expressed circulating miRNAs asbiomarkers for endometriosis, was demonstrated. The results demonstratedthat miR-125b-5p expression had the highest AUC levels among the miRNAsidentified in the microarray data. Endometriosis is associated with a6.7 year average diagnostic delay, resulting in progression of thedisease and impairment in quality of life (Nnoaham et al., 2011, FertilSteril, 96:366-373). Early diagnosis of endometriosis would allow prompttreatment, improvement in quality of life and potentially preservationof fertility. Several growth factors and cytokines have been studied inserum, plasma and urine as potential biomarkers of this disease (Cho etal., 2012, Hum Reprod, 27:515-522, Reis et al., 2012, Hum Reprod,27:1445-1450); however, none of these are sufficiently sensitive orspecific to be translated into a clinical diagnostic tool forendometriosis (May et al., 2010, Hum Reprod, 16:651-674).

There is little known about the mechanisms by which miRNAs aretransported to plasma and their biological impact on distant organs.Normal endometrium as well as paired eutopic and ectopic endometriumsamples have differential miRNA expression patterns (Jia et al., 2013,Hum Reprod, 28:322-330). Furthermore, a strong association between themiRNA profiles of the serum/plasma and tissues has been demonstrated instudies examining several cancers (Resnick et al., 2009, Gynecol Oncol,112:55-59, Wang et al., 2013, J Clin Endocrinol Metab, 98:281-289). Inaddition to their role as biomarkers, circulating miRNAs have aregulatory effect on distant tissues. The circulating miRNAs identifiedhere is a means of communication between endometriosis and endometrium.Further, they have systemic effects, altering distant organ systems. Ithas been demonstrated in an animal model that endometriosis far removedfrom the peritoneal cavity affects uterine gene expression (Naqvi etal., 2016, Reprod Sci, 23:186-191). Circulating miRNAs is a signalingmechanism by which endometriosis has an effect on the uterus.

It has been demonstrated that miRNAs contribute to the pathogenesis ofendometriosis by regulating the abnormal cell differentiation, invasionand inflammation (Petracco et al., 2011, J Clin Endocrinol Metab,96:E1925-E1933, Teague et al., 2010, Hum Reprod Update, 16:142-165,Naqvi et al., 2016, Reprod Sci, 23:186-191). It has also beendemonstrated that many individual circulating miRNAs are identified inendometriosis (Jia et al., 2013, Hum Reprod, 28:322-330, Wang et al.,2013, J Clin Endocrinol Metab, 98:281-289, Cho et al., 2015, FertilSteril, 103(5):1252-1260). Jia et al. reported that plasma miR-17-5p,miR-20a and miR-22 were down-regulated in women with endometriosis (Jiaet al., 2013, Hum Reprod, 28:322-330), however these miRNAs did notreach the cut off threshold and was not examined further in this study.The differences in miRNAs identified are due to the differences in age,race disease severity or use of medical therapies.

In the first comprehensive analysis of all miRNA in the circulation ofwomen with endometriosis it was identified that miR-125b-5p is the bestsingle candidate biomarker of endometriosis. miR-125b-5p wassignificantly upregulated in serum of women with endometriosis and ithad the highest AUC as well as sensitivity and specificity (AUC=0.974,95% Confidence Interval [CI], 0.00-1.00, p<0.001) (100% and 96%respectively) of any single miRNA. miR-125b-5p has demonstrated a rolein invasion and the pathogenesis of cancer and acts as a oncogene inlung cancer (Wang et al., 2015, Mol Med Rep, 11(5):3880-3887). It isupregulated in the serum of breast cancer patients and is associatedwith chemotherapeutic resistance, nonresponsive patients having higherexpression levels (Wang et al., 2012, PLoS One, 7:e34210, Matamala etal., 2015, Clin Chem, 61(8):1098-1106), while significantly higherlevels found in the serum of HBV positive hepatocellular carcinomapatients (Giray et al., 2014, Mol Biol Rep, 41(7):4513-4519). It hasalso been demonstrated that miR-125b-5p regulates osteogenicdifferentiation of hBMSCs through the modulation of Osx expression (Chenet al., 2014, Mol Med Rep, 9(5):1820-1826). In this study, majority ofcases had severe endometriosis with invasion and miRNA 125, whichcontributes to that phenotype. Other miRNAs found in this study alsocontribute to the etiology of this disease. miR-451a has tumorsuppressing function in neuroblastoma and osteosarcoma (Liu et al.,2016, Mol Med Rep, doi: 10.3892/mmr. 4770, Xu et al., 2014, Cell BiochemBiophys, 69(1):163-168) and elevated levels found in endometriotictissue that function to limit endometriotic lesion cell survival (Grahamet al., 2015, Hum Reprod, 30(3):642-652).

In conclusion, this study showed the differential expression ofcirculating miRNAs, particularly miR-125b-5p, in endometriosis. Thefindings also support the clinical utility of plasma miRNA profiling inthe diagnosis of this disease. Given the lack of biomarkers of diseaseand the delay in diagnosis, miRNA is useful in early detection andintervention. Further, the role of circulating miRNAs provides a betterunderstanding of the systemic effects of endometriosis and allow formore novel treatments.

AUC, 95% Confidence Interval (CI) and P values of the differentiallyexpressed miRNAs miRNAs AUC 95% CI p-value miR-125b-5p 0.974 0.000-1.000<0.001 miR-150-5p 0.808 0.680-0.936 <0.001 miR-342-3p 0.760 0.608-0.9120.002 miR-143-3p 0.926 0.000-1.000 <0.001 miR-500a-3p 0.901 0.803-0.998<0.001 miR-451a 0.835 0.707-0.963 <0.001 miR-18a-5p 0.797 0.653-0.9400.001 miR-6755-3p 0.718 0.577-0.860 0.008 miR-3613-5p 0.862 0.740-0.985<0.001

TABLE 3 Top Hits Microarray Data miR-125b-5p 77.07 E1 up vs C1miR-214-3p 62.5954 E1 up vs C1 miR-150-5p 57.2203 E1 up vs C1 miR-342-3p41.5546 E1 up vs C1 miR-145-5p 38.1178 E1 up vs C1 miR-143-3p 29.4994 E1up vs C1 miR-126-3p 27.3483 E1 up vs C1 miR-500a-3p 18.0946 E1 up vs C1miR-451a 16.8822 E1 up vs C1 miR-18a-5p 14.4632 E1 up vs C1 miR-6755-3p−12.4883 E1 down vs C1 miR-553 −18.6982 E1 down vs C1 miR-3613-5p−102.613 E1 down vs C1 miR-4668-3p −124.901 E1 down vs C1

Example 2: Relative Expression of miRNAs in Endometriosis Lesions inBaboons

The relative expression of miRNAs in endometriotic lesions in Baboons asdetermined by real time quantitative PCR as shown in FIG. 4. Thecomparison of the relative expression levels of miR-125b-5p, miR-150-5pand miR-3613-5p with the relative expression levels of the respectivemiRNAs after statin treatment was performed. It was observed that thelesions shrunk in the statin treated animals. The miR-125b-5p expressionlevels in samples of untreated animals were shown to have higherrelative expression levels than samples that were taken from treatedanimals. Further, it was observed that the relative level of miR-3613-5pwas lower in samples from control untreated animals in comparison to thelevel of miR-3613-5p in samples from treated animals. Lastly, it wasobserved that the relative level of miR-150-5p was lower in samples fromcontrol untreated animals, in comparison to the level of miR-150-5p insamples from treated animals.

Example 3: Saliva MicroRNAs as Diagnostic Markers for Endometriosis

Step 1: RNA Extraction from Saliva

Saliva samples (200 μL) were collected and transferred to 1.5 mL tubes.RNase free water was added to samples with volumes less than 200 μL inorder to bring the total sample volume to 200 μL. 1 mL of QIAzol LysisReagent (Qiagen) was added to the sample. The tube was vortexed briefly,and the sample was allowed to incubate at room temperature for fiveminutes. Then, 200 μL of choloroform was added to the lysate andvortexed for approximately 15 seconds. The sample mixture was thenincubated for two minutes at room temperature and centrifuged at12,000×g for fifteen minutes in a cold room (approximately 4° C.).Approximately 560 μL of the aqueous phase was transferred to a new 1.5mL tube. 840 μL of 100% ethanol was added to the 560 μL of aqueous phaseto obtain a total volume of 1400 μL. 700 μL of the mixture was thentransferred into a RNeasy MiniElute spin column with 2 mL collectiontube. The spin column with collection tube was centrifuged at 9,000×gfor 15 seconds. The flow-through was discarded and the remaining 700 μLof mixture was transferred to the spin column with collection tube andagain centrifuged at 9,000×g for 15 seconds. The flow-through wasdiscarded and 700 μL of buffer RWT was added to each spin column andthen centrifuged at 9,000×g for 15 seconds. The flow-through wasdiscarded and 500 μL of buffer RPE was added to each spin column andthen centrifuged at 9,000×g for 15 seconds. The flow-through wasdiscarded and another 500 μL of buffer RPE was added to the spin columnand again centrifuged at 9,000×g for 15 seconds. Then, 500 μL of 80%ethanol was added to the spin column and centrifuged at 9,000×g for 2minutes. The flow-through and collection tube were subsequentlydiscarded and the spin column was transferred to a new 2 mL collectiontube. The lid of the spin column was left open and then centrifuged at12,000×g for 5 minutes to dry the membrane. The spin column was thenplaced in a 1.5 mL tube. 14 μL of RNase-free water was added to the spincolumn and it was centrifuged at 12,000×g for 1 minute to elute totalRNA. The spin column was discarded and the RNA was stored at −80° C.

Step 2: Preparation of cDNA

The TaqMan Advanced miRNA cDNA Synthesis Kit was used to prepare cDNA(ThermoFisher, catalog number: A28007) in four sequential steps: A, B,C, and D.

Step A was performed with the following contents in each reaction: 0.5μL 10× Poly A buffer; 0.5 μL 10 mM ATP; 0.3 μL Poly A enzyme, 5 U/μL;1.7 μL RNase-free water; 2.0 μL sample. The plate or tube was sealed andvortexed briefly. The plate or tube was centrifuged to spin down thecontents and eliminate any air bubbles. The plate or tube was placedinto a thermal cycler and incubated with the following settings:

1. Polyadenylation at 37° C. for 45 minutes.

2. Stop reaction at 65° C. for 10 minutes.

3. Hold at 4° C.

Step B was performed with the following contents in each reaction: 3.0μL 5×DNA Ligase Buffer; 4.5 μL 50% PEG 8000; 0.6 μL 25× LigationAdaptor; 1.5 μL RNA Ligase; 0.4 μL RNase-free water. The LigationReaction Mix was vortexed to thoroughly mix the contents and thencentrifuged briefly to spin down the contents and eliminate air bubbles.10 μL of the Ligation Reaction Mix was transferred to each well of thereaction plate or each reaction tube containing the poly(A) tailingreaction product. The reaction plate or tubes were sealed, then vortexedbriefly or shaken (1,900 RPM for 1 minute with an Eppendorf™ MixMate™)to thoroughly mix the contents. The reaction plate or tubes werecentrifuged briefly to spin down the contents. The plate or tube wasplaced into the thermal cycler.

Step C was performed with the following contents in each reaction: 6 μL5×RT Buffer; 1.2 μL dNTP Mix (25 mM each); 1.5 μL 20× Universal RTPrimer; 3 μL 10×RT Enzyme Mix; 3.3 μL RNase-free water. The RT ReactionMix was vortexed to thoroughly mix the contents, then centrifugedbriefly to spin down the contents and eliminate air bubbles. 15 μl ofthe RT Reaction Mix was transferred to each well of the reaction plateor each reaction tube containing the adaptor ligation reaction product.The total volume was 30 μl per well or tube. The reaction plate or tubeswere then sealed and vortexed briefly to thoroughly mix the contents.The reaction plate or tubes were then centrifuged briefly to spin downthe contents. The plate or tube was placed in the thermal cycler andincubated with the following settings:

1. Reverse transcription at 42° C. for 15 minutes

2. Stop reaction at 85° C. for 5 minutes

3. Hold at 4° C.

Step D was performed with the following contents in each reaction: 25 μL2×miR-Amp Master Mix; 2.5 μL 20×miR-Amp Primer Mix; 17.5 μL RNase-freewater. The miR-Amp Reaction Mix was vortexed to thoroughly mix thecontents, then centrifuged briefly to spin down the contents andeliminate air bubbles. 45 μL of the miR-Amp Reaction Mix was transferredto each well of a new reaction plate or reaction tube. 5 μL of the RTreaction product was added to each reaction well or reaction tube. Thetotal volume in each well or tube was 50 μL. The reaction plate or tubeswere sealed and then vortexed briefly to thoroughly mix the contents.The reaction plate or tubes were then briefly centrifuged to spin downto mix the contents. The reaction plate or tubes were placed into athermal cycler and then incubated using the following settings, MAX rampspeed, and standard cycling:

1. Enzyme activation at 95° C. for 5 minutes, 1 cycle

2. Denature at 95° C. for 3 seconds, 14 cycles

3. Anneal/Extend at 60° C. for 30 seconds, 14 cycles

4. Stop reaction at 99° C. for 10 minutes, 1 cycle.

5. Hold at 4° C.

Step 3: Amplification of microRNAs

RT-PCR Protocol:

1. 95° C. for 3 min

2. 95° C. for 15 s

3. 59° C. for 5 s

4. 72° C. for 55 s

5. Repeat 2-4 steps for 39 cycles

6. Melting Curve at 55° C. for 10 s

7. 95° C. for 5 s

8. Hold at 4° C.

The relative expression of saliva miRNAs that were differentiallyexpressed between the control group and the endometriosis group areshown in FIG. 5 and FIG. 6. The data represents fifteen samples in eachgroup. The fold change for miR 125b-5p, Let7b and miR150-5p are 25, 7and 1.8 fold respectively. These fold changes are statisticallysignificant when comparing the endometriosis group to the control groupas the p-value is less than 0.05 (p<0.05) for the above miRs. Thep-values are as follows: miR 125b-5p (p<0.001), Let7b (p<0.025) and miR150-5p (p<0.047). A statistically-significant downregulation ofmiR-3613-5p was observed.

Example 4: Serum MicroRNAs Used to Diagnose Endometriosis Prior toSurgical Diagnosis

Reproductive age women undergoing laparoscopy or laparotomy for benigngynecologic indication were enrolled in a prospective case controlstudy. Patients were stratified into the disease group if surgeryconfirmed presence of endometriosis, and control if surgery revealedother benign pathology. Twenty-three control patients and seventeenendometriosis patients were enrolled and met inclusion criteria in thestudy. Controls included 4 patients with dermoid cysts, 3 withcystadenomas, 6 with fibroids, 2 with evidence of chronic pelvicinfection, and 8 without abnormal pathology. Expression levels ofmicroRNAs 125b and 150 were significantly higher in patients withendometriosis compared to control (microRNA 125: X vs Y; p=0.028,MicroRNA 150: Z vs W; p=0.014, respectively). 4 patients with pelvicpain and suspected endometriosis were not found to have endometriosis onsurgical evaluation. Expression of 125b and 150 in these patients wasconsistent with that of controls, and differed from that of patientswith surgically confirmed endometriosis.

Total microRNA was extracted from serum using a miRNeasy mini kit,(Qiagen) following manufacturer's protocol. Preparation of cDNA wasperformed using TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher).Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was carriedout to determine the expression levels of microRNAs 125b-5p and 150-5p.The expression levels of the serum microRNAs between the control andendometriosis group were compared using the Mann-Whitney U test. Resultsof the study (FIG. 7) showed that microRNAs 125b-5p and 150-5p weresignificantly elevated in patients with endometriosis compared tocontrols.

Example 5: Diagnosis and Treatment of Endometriosis

In this prophetic example, a blood or saliva sample is taken from afemale patient with symptoms of endometriosis. The quantity of microRNA125b-5p is then determined in the sample, and the patient is diagnosedwith endometriosis if the quantity is above a threshold value. Thepatient is treated with a therapeutically effective dose of a compound.The compound is given to the patient by oral, intravenous, orintramuscular administration. The compound causes a reduction in thesymptoms of endometriosis. The patient is treated about once everymonth. After one month of treatment, six months of treatment, and oneyear of treatment, the patient is assessed for reduction in symptoms ofendometriosis.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the disclosure describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of diagnosing a subject suspected ofhaving endometriosis comprising: a. providing a saliva, sputum, urine,lymphatic fluid, synovial fluid, cerebrospinal fluid, stool, or mucussample from the subject, wherein the saliva, sputum, urine, lymphaticfluid, synovial fluid, cerebrospinal fluid, stool, or mucus samplecomprises miRNA associated with endometriosis; b. detecting a level ofthe miRNA associated with endometriosis; c. comparing the detected levelof miRNA associated with endometriosis with a reference value in orderto determine a relative level of miRNA associated with endometriosis inthe saliva, sputum, urine, lymphatic fluid, synovial fluid,cerebrospinal fluid, stool, or mucus sample; and d. diagnosing thesubject with endometriosis based on the relative level of miRNAassociated with endometriosis in the saliva, sputum, urine, lymphaticfluid, synovial fluid, cerebrospinal fluid, stool, or mucus sample,wherein the diagnosing the subject with endometriosis has a specificityor sensitivity greater than 90%.
 2. A method detecting miRNA comprising:a. providing a saliva, sputum, urine, lymphatic fluid, synovial fluid,cerebrospinal fluid, stool, or mucus sample from a subject, wherein thesample comprises nucleic acids and wherein the subject is suspected ofhaving endometriosis; b. performing an amplification or sequencingreaction on the nucleic acids in order to detect a presence of at leastone miRNA in the sample, wherein the at least one miRNA is selected fromthe group consisting of miR-18, miR-125, miR-126, miR-143, miR-145,miR-150, miR-214, miR-342, miR-451, miR-500, miR-553, miR-3613,miR-4668, and miR-6755; and c. comparing the presence of the miRNA to areference value.
 3. The method of any of the preceding claims, whereinthe sample is a fluid.
 4. The method of any of the preceding claims,wherein the sample is blood, plasma, or serum.
 5. The method of any ofthe preceding claims, wherein the sample is saliva.
 6. The method of anyof the preceding claims, wherein the sample is cell-free.
 7. The methodof any of the preceding claims, wherein the reference value is thepresence of the at least one miRNA in samples from subjects that do nothave endometriosis.
 8. The method of any of the preceding claims,wherein the miRNA is selected from the group consisting of miR-125,miR-451, and miR-3613.
 9. The method of any of the preceding claims,wherein the miRNA is miR-125b.
 10. The method of any of the precedingclaims, wherein the miRNA is miR-125b-5p.
 11. The method of any of thepreceding claims, wherein the miRNA is miR-451a.
 12. The method of anyof the preceding claims, wherein the miRNA is miR-3613-5p.
 13. A methodcomprising: a. providing a sample from a subject, wherein the samplecomprises nucleic acids and wherein the subject is suspected of havingendometriosis; b. performing sequencing reaction on the nucleic acids todetect a presence of at least one miRNA in the sample, wherein the atleast one miRNA is selected from the group consisting of miR-18,miR-125, miR-126, miR-143, miR-145, miR-150, miR-214, miR-342, miR-451,miR-500, miR-553, miR-3613, miR-4668, and miR-6755; and c. comparing thepresence of the miRNA to a reference value.
 14. The method of any one ofthe preceding claims, wherein the at least one miRNA is detected byhigh-throughput or massively-parallel sequencing.
 15. A method ofdiagnosing a subject exhibiting symptoms of endometriosis or suspectedof having endometriosis, the method comprising: a. detecting a presenceof at least one miRNA that is not let-7, miR-135, miR-449a, miR-34c,miR-200a, miR-200b, miR-141, miR-125b-5p, miR-150-5p, miR-342-3p,miR-145-5p, miR-143-3p, miR-500a-3p, miR-451a, miR-18a-5p, miR-6755-3p,miR-3613-5p; and b. diagnosing the subject with endometriosis, when thedetected miRNA level is above a threshold level, wherein the diagnosingthe subject with endometriosis has a specificity or sensitivity greaterthan 90%.
 16. The method of any of the preceding claims, wherein themiRNA is cell-free.
 17. The method of any of the preceding claims,wherein the diagnosing the subject with endometriosis is diagnosing thesubject with early stage endometriosis.
 18. The method of any of thepreceding claims, wherein the diagnosing the subject with endometriosisis diagnosing the subject with moderate-to-severe disease.
 19. Themethod of any of the preceding claims, wherein the diagnosing thesubject with endometriosis is diagnosing the subject with stage III orStage IV endometriosis.
 20. The method of any of the preceding claims,wherein the diagnosing the subject with endometriosis further comprisesmonitoring the subject for changes in miRNA expression levels followingtreatment.
 21. The method of any of the preceding claims, wherein thediagnosing the subject with endometriosis has a specificity orsensitivity greater than 85%.
 22. The method of any of the precedingclaims, wherein the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%.
 23. The method of any ofthe preceding claims, wherein the method further comprises treating thesubject for endometriosis.
 24. The method of any of the precedingclaims, wherein treatment is a medication to relieve pain.
 25. Themethod of any of the preceding claims, wherein treatment is hormonetherapy.
 26. The method of any of the preceding claims, whereintreatment is a hormonal contraceptive.
 27. The method of any of thepreceding claims, wherein treatment is a Gonadotropin-releasing hormoneagonist.
 28. The method of any of the preceding claims, whereintreatment is a Gonadotropin-releasing hormone antagonist.
 29. The methodof any of the preceding claims, wherein the detecting comprises: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with a probe specific for the at least one miRNA, whereinthe probe emits a signal upon binding the produced cDNA; and (iii) usinga detector to detect the signal emitted by the probe.
 30. A method ofdetecting miRNA comprising: a. providing a sample from a subject,wherein the sample comprises nucleic acids and wherein the subject issuspected of having endometriosis; b. performing an amplification,microarray or sequencing reaction on the nucleic acids; c. detecting apresence of at least one miRNA in the sample, wherein the at least onemiRNA is selected from the group consisting of miR-126, miR-214,miR-553, and miR-4668; and d. comparing the presence of the miRNA to areference value.
 31. The method of any of the preceding claims, whereinthe sample is a fluid.
 32. The method of any of the preceding claims,wherein the sample is blood, plasma, saliva, or serum.
 33. The method ofany of the preceding claims, wherein the reference value is the presenceof the at least one miRNA in samples from subjects that do not haveendometriosis.
 34. The method of any of the preceding claims, whereinthe detecting is by polymerase chain reaction (PCR).
 35. The method ofany of the preceding claims, wherein the detecting is by quantitativePCR.
 36. The method of any of the preceding claims, wherein thedetecting comprises hybridizing a unique primer to the at least onemiRNA or to cDNA derived from the at least one miRNA.
 37. The method ofany of the preceding claims, wherein the detecting further comprisesperforming a reverse transcription reaction on the at least one miRNAusing at least one primer or probe specific for the at least one miRNAor using at least one universal primer.
 38. The method of any of thepreceding claims, wherein the detecting comprises: (i) amplifying miRNAwithin the sample by performing a reverse transcription assay using aprimer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with an intercalating dye that emits a signal; and (iii) using adetector to detect the emitted signal over time.
 39. The method of anyof the preceding claims, wherein the detecting comprises: (i) amplifyingmiRNA within the sample by performing a reverse transcription assayusing a primer that specifically binds the at least one miRNA or using auniversal primer, thereby producing cDNA; (ii) contacting the producedcDNA with a probe specific for the at least one miRNA, wherein the probeemits a signal upon binding the produced cDNA; and (iii) using adetector to detect the signal emitted by the probe.
 40. The method ofthe preceding claim, wherein the probe is attached to a fluorophore anda quencher.
 41. The method of any of the preceding claims, wherein thedetecting is by sequencing.
 42. The method of any of the precedingclaims, wherein the endometriosis is diagnosed when the at least onemiRNA is at least 2-fold greater than the reference value.
 43. Themethod of any of the preceding claims, wherein the endometriosis isdiagnosed when the at least one miRNA is at least 2-fold less than thereference value.
 44. The method of any of the preceding claims, whereinthe endometriosis is diagnosed when miRNA-125b-5p is upregulated,miR-150-5p is upregulated, miR-342-3p is upregulated, miR-145-5p isupregulated, miR-143-3p is upregulated, miR-500a-3p is upregulated, ormiR-18a-5p is upregulated,
 45. The method of any of the precedingclaims, wherein the endometriosis is diagnosed when at least three miRNAare upregulated wherein the at least three miRNA are from the groupconsisting of miRNA-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, and miR-18a-5p.
 46. The method of any of thepreceding claims, wherein the endometriosis is diagnosed whenmiR-6755-3p is downregulated or miR-3613-5p is downregulated.
 47. Themethod of any of the preceding claims, wherein the endometriosis isdiagnosed when miR-125b-5p is upregulated, miR-451a is upregulated, andmiR-3613-5p is downregulated.
 48. The method of any of the precedingclaims, wherein the endometriosis is diagnosed when miR-125b-5p isupregulated.
 49. The method of any of the preceding claims, wherein thesubject is negative for the presence of a KRAS variant allele.
 50. Themethod of any of the preceding claims, wherein the detected miRNA levelis used to determine severity of disease.
 51. The method of any of thepreceding claims, wherein the subject is a human subject.
 52. A methodof treating a subject with endometriosis, comprising administering anendometriosis treatment to a subject identified as having a differentiallevel of at least one miRNA selected from the group consisting ofmiR-18a-5p, miR-125, miR-126, miR-143, miR-145, miR-150-5p, miR-214,miR-342, miR-451, miR-500, miR-553, miR-3613, miR-4668, and miR-6755 ina biological sample of the subject as compared to a comparator.
 53. Themethod of claim 52, further comprising diagnosing the subject withendometriosis when the subject is identified as having a differentiallevel of at least one miRNA selected from the group consisting ofmiR-18a-5p, miR-125, miR-126, miR-143, miR-145, miR-150-5p, miR-214,miR-342, miR-451, miR-500, miR-553, miR-3613, miR-4668, and miR-6755, ascompared to the comparator.
 54. The method of claim 53, wherein thediagnosing the subject with endometriosis comprises providing abiological sample from the subject and detecting in the biologicalsample the differential level of at least one miRNA selected from thegroup consisting of the miR-18a-5p, miR-125, miR-126, miR-143, miR-145,miR-150-5p, miR-214, miR-342, miR-451, miR-500, miR-553, miR-3613,miR-4668, and miR-6755, as compared to the comparator.
 55. The method ofclaim 54, wherein the biological sample is a body fluid.
 56. The methodof claim 55, wherein the body fluid is blood, plasma, or serum.
 57. Themethod of claim 55, wherein the body fluid is saliva.
 58. The method ofclaim 55, wherein the body fluid is urine.
 59. A method of diagnosingand treating a subject suspected of having endometriosis comprising: a.providing a saliva, sputum, urine, lymphatic fluid, synovial fluid,cerebrospinal fluid, stool, or mucus sample from the subject, whereinthe sample comprises miRNA associated with endometriosis; b. detecting alevel of the miRNA associated with endometriosis; c. comparing thedetected level of miRNA associated with endometriosis with a referencevalue in order to determine a relative level of miRNA associated withendometriosis in the fluid sample; d. diagnosing the subject withendometriosis based on the relative level of miRNA associated withendometriosis in the saliva, sputum, urine, lymphatic fluid, synovialfluid, cerebrospinal fluid, stool or mucus sample, wherein thediagnosing the subject with endometriosis has a sensitivity orspecificity greater than 90%; and e. administering a treatment to thesubject diagnosed with endometriosis
 60. A method of diagnosing andtreating a subject suspected of having endometriosis, the methodcomprising: a. detecting a presence of at least one miRNA that is notlet-7, miR-449a, miR-34c, miR-200a, miR-200b, miR-150, miR-18a, ormiR-141; b. diagnosing the subject with endometriosis, when the detectedmiRNA level is above a threshold level, wherein the diagnosing thesubject with endometriosis has a specificity or sensitivity greater than90%; and c. administering a treatment to the subject diagnosed withendometriosis.
 61. The method of any one of the preceding claims,wherein the at least one miRNA comprises miR-125b-5p, miR-451a, andmiR-3613-5p.
 62. The method of any one of the preceding claims, whereinthe at least one miRNA is miR-125b-5p,
 63. The method of any one of thepreceding claims, wherein the at least one miRNA is miR-3613-5p.
 64. Themethod of any one of the preceding claims, wherein the at least onemiRNA is cell-free miRNA.
 65. The method of any one of the precedingclaims, wherein the at least one miRNA is present in at least a 2-foldchange compared to the comparator.
 66. The method of any one of thepreceding claims, wherein the diagnosing the subject with endometriosisis diagnosing the subject with early stage endometriosis.
 67. The methodof any one of the preceding claims, wherein the diagnosing the subjectwith endometriosis is diagnosing the subject with moderate-to-severedisease.
 68. The method of any one of the preceding claims, wherein thediagnosing the subject with endometriosis is diagnosing the subject withstage III or stage IV endometriosis.
 69. The method of any one of thepreceding claims, wherein the diagnosing the subject with endometriosisfurther comprises monitoring the subject for changes in miRNA expressionlevels following treatment.
 70. The method of any one of the precedingclaims, wherein the diagnosing the subject with endometriosis has aspecificity or sensitivity greater than 95%.
 71. The method of any oneof the preceding claims, wherein
 72. The method of any one of thepreceding claims, wherein the treatment is a medication to relieve pain.73. The method of claim 1, wherein treatment is selected from the groupconsisting of a hormone therapy, hormonal contraceptive, aGonadotropin-releasing hormone agonist, and a Gonadotropin-releasinghormone antagonist.
 74. The method of claim 2, further comprisingdetecting the at least one miRNA by performing a polymerase chainreaction (PCR).
 75. The method of claim 2, further comprising detectingthe at least one miRNA by performing quantitative PCR.
 76. The method ofclaim 2, further comprising detecting the at least one miRNA by: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with an intercalating dye that emits a signal; and (iii)using a detector to detect the emitted signal over time.
 77. The methodof claim 2, further comprising detecting the at least one miRNA by: (i)amplifying miRNA within the sample by performing a reverse transcriptionassay using a primer that specifically binds the at least one miRNA orusing a universal primer, thereby producing cDNA; (ii) contacting theproduced cDNA with a probe specific for the at least one miRNA, whereinthe probe emits a signal upon binding the produced cDNA; and (iii) usinga detector to detect the signal emitted by the probe.
 78. The method ofclaim 2, further comprising detecting the at least one miRNA bysequencing.
 79. The method of claim 2, further comprising detecting theat least one miRNA by high-throughput sequencing or massively parallelsequencing.
 80. The method of claim 2, wherein the at least one miRNA ispresent at a level at least 2-fold greater than the reference value. 81.The method of claim 1, wherein the subject is negative for the presenceof KRAS variant allele.
 82. The method of claim 1, wherein a level ofthe at least one miRNA is used to determine severity of disease.
 83. Themethod of claim 1 or claim 3, wherein the subject is a human subject.84. The method of claim 2, wherein the endometriosis is diagnosed whenmiRNA-125b-5p is upregulated, miR-150-5p is upregulated, miR-342-3p isupregulated, miR-145-5p is upregulated, miR-143-3p is upregulated,miR-500a-3p is upregulated, or miR-18a-5p is upregulated,
 85. The methodof claim 2, wherein the endometriosis is diagnosed when at least threemiRNA are upregulated wherein the at least three miRNA are from thegroup consisting of miRNA-125b-5p, miR-150-5p, miR-342-3p, miR-145-5p,miR-143-3p, miR-500a-3p, and miR-18a-5p.
 86. The method of claim 2,wherein the endometriosis is diagnosed when miR-6755-3p is downregulatedor miR-3613-5p is downregulated.
 87. The method of claim 2, wherein theendometriosis is diagnosed when miR-125b-5p is upregulated, miR-451a isupregulated, and miR-3613-5p is downregulated.
 88. The method of claim2, wherein the endometriosis is diagnosed when miR-125b-5p isupregulated.
 89. A method for detecting microRNA (“miRNA”) in a samplecomprising: a. providing a saliva, sputum, urine, lymphatic fluid,synovial fluid, cerebrospinal fluid, stool, or mucus sample from asubject, wherein the sample comprises nucleic acids and wherein thesubject is suspected of having endometriosis; b. performing anamplification or sequencing reaction on the nucleic acids; c. detectinga presence of at least one miRNA in the sample, wherein the at least onemiRNA is selected from the group consisting of miR-18, miR-125, miR-126,miR-143, miR-145, miR-150, miR-214, miR-342, miR-451, miR-500, miR-553,miR-3613, miR-4668, and miR-6755; and d. comparing the presence of themiRNA to a reference value.
 90. A method of diagnosing endometriosis ina subject, the method comprising: a. determining the level of at leastone miRNA in a biological sample of the subject, wherein the miRNA is atleast one selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668, and b. comparing the level ofthe at least one miRNA in the biological sample with the level of the atleast one miRNA in a comparator, wherein when the level of the at leastone miRNA in the biological sample is different than the level of the atleast one miRNA in the comparator, the subject is diagnosed withendometriosis.
 91. The method of any of the preceding claims, whereinthe at least one miRNA is the combination of miR-125, miR-451 andmiR-3613.
 92. The method of any of the preceding claims, wherein thesubject is human.
 93. The method of any of the preceding claims, furthercomprising the step of treating the subject for endometriosis.
 94. Themethod of any of the preceding claims, wherein the comparator is atleast one comparator selected from the group consisting of a positivecontrol, a negative control, a normal control, a wild-type control, ahistorical control, and a historical norm.
 95. The method of any of thepreceding claims, wherein the method comprises detecting that the levelof at least one of miR-125, miR-150, miR-342, miR-145, miR-143, miR-500,miR-451, miR-18, miR-214, and miR-126 is increased in the biologicalsample compared to the level in the comparator.
 96. The method of any ofthe preceding claims, wherein the method comprises detecting that thelevel of at least one of miR-6755, miR-3613, miR-553, and miR-4668 aredecreased in the biological sample compared to the level in thecomparator.
 97. The method of any of the preceding claims, whereindetermining the level of the at least one miRNA utilizes at least onetechnique selected from the group consisting of reverse transcription,PCR, and a microarray.
 98. The method of any of the preceding claims,wherein the biological sample is selected from the group consisting ofblood, serum, plasma, and any a combination thereof.
 99. A method ofdiagnosing or providing a prognosis for endometriosis in a subject, themethod comprising the step of: detecting altered expression of at leastone miRNA selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668 in a biological sample of thesubject suspected of or having endometriosis.
 100. The method of any oneof the preceding claims, wherein the at least one miRNA is thecombination of miR-125, miR-451, and miR-3613.
 101. The method of anyone of the preceding claims, wherein the biological sample is selectedfrom the group consisting of blood, serum, plasma, and any combinationthereof.
 102. A kit comprising a reagent that selectively binds to atleast one miRNA, wherein the at least one miRNA is at least one selectedfrom the group consisting of miR-125, miR-150, miR-342, miR-145,miR-143, miR-500, miR-451, miR-18, miR-214, miR-126, miR-6755, miR-3613,miR-553, and miR-4668.
 103. The kit of claim 97, comprising at leastthree reagents, wherein the first reagent selectively binds to miR-125,wherein the second reagent selectively binds to miR-451 and wherein thethird reagent selectively binds to miR-3613.
 104. A method of treating asubject with endometriosis, comprising administering an endometriosistreatment to a subject identified as having a differential level of atleast one miRNA selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668 in a biological sample of thesubject as compared to a comparator.
 105. A method of monitoring aresponse to an endometriosis treatment in a subject being treated forendometriosis, the method comprising: a. determining the level of atleast one miRNA in a biological sample of the subject, wherein the miRNAis at least one selected from the group consisting of miR-125, miR-150,miR-342, miR-145, miR-143, miR-500, miR-451, miR-18, miR-214, miR-126,miR-6755, miR-3613, miR-553, and miR-4668, and b. comparing the level ofthe at least one miRNA in the biological sample with the level of the atleast one miRNA in a comparator.
 106. The method of any of the precedingclaims wherein the at least one miRNA comprises let-7, let-7a, let-7b,let-7c, let-7d, let-7e, let-7f, or let-7g.